Treatment of alpha-galactosidase a deficiency

ABSTRACT

The invention provides highly purified α-Gal A, and various methods for purifying it; α-Gal A preparations with altered charge and methods for making those preparations; α-Gal A preparations that have an extended circulating half-life in a mammalian host, and methods for making same; and methods and dosages for administering an α-Gal A preparation to a subject.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.08/928,881, filed on Sep. 13, 1996, and PCT/US97/16603, filed on Sep.12, 1997, which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of α-galactosidase A deficiency.

BACKGROUND OF THE INVENTION

Fabry disease is an X-linked inherited lysosomal storage diseasecharacterized by severe renal impairment, angiokeratomas, andcardiovascular abnormalities, including ventricular enlargement andmitral valve insufficiency. Fabry disease also affects the peripheralnervous system, causing episodes of agonizing, burning pain in theextremities. Fabry disease is caused by a deficiency in the enzymeα-galactosidase A (α-Gal A). α-Gal A is the lysosomal glycohydrolasethat cleaves the terminal α-galactosyl moieties of variousglycoconjugates. Fabry disease results in a blockage of the catabolismof the neutral glycosphingolipid, ceramide trihexoside (CTH), andaccumulation of this enzyme substrate within cells and in thebloodstream.

Due to the X-linked inheritance pattern of the disease, most Fabrydisease patients are male. Although severely affected femaleheterozygotes have been observed, female heterozygotes are oftenasymptomatic or have relatively mild symptoms (such as a characteristicopacity of the cornea). An atypical variant of Fabry disease, exhibitinglow residual α-Gal A activity and either very mild symptoms orapparently no other symptoms characteristic of Fabry disease, correlateswith left ventricular hypertrophy and cardiac disease. Nakano et al.,New Engl. J. Med. 333: 288-293 (1995). A reduction in α-Gal A may be thecause of such cardiac abnormalities.

The cDNA and gene encoding human α-Gal A have been isolated andsequenced. Human α-Gal A is expressed as a 429-amino acid polypeptide,of which the N-terminal 31 amino acids are the signal peptide. The humanenzyme has been expressed in Chinese Hamster Ovary (CHO) cells (Desnicket al., U.S. Pat. No. 5,356,804; Ioannou et al., J. Cell Biol. 119: 1137(1992)); and insect cells (Calhoun et al., WO 90/11353).

However, current preparations of α-Gal A have limited efficacy. Methodsfor the preparation of α-Gal A with relatively high purity depend on theuse of affinity chromatography, using a combination of lectin affinitychromatography (concanavalin A (Con A) Sepharose®) and affinitychromatography based on binding of α-Gal A to the substrate analogN-6-aminohexanoyl-α-D-galactosylamine coupled to a Sepharose® matrix.See, e.g., Bishop et al., J. Biol. Chem. 256:1307-1316 (1981). The useof proteinaceous lectin affinity resins and substrate analog resins istypically associated with the continuous leaching of the affinity agentfrom the solid support (Marikar et al., Anal. Biochem. 201: 306-310(1992), resulting in contamination of the purified product with theaffinity agent either free in solution or bound to eluted protein. Suchcontaminants make the product unsuitable for use in pharmaceuticalpreparations. Bound substrate analogs and lectins can also havesubstantial negative effects on the enzymatic, functional, andstructural properties of proteins. Moreover, α-Gal A produced by themethods in the prior art is rapidly eliminated by the liver.

Thus, a need remains in the art for a purification protocol usingconventional chromatography resins, which are readily available insupplies and quality suitable for large-scale commercial use, and whichproduces an α-Gal A preparation that is free of affinity agent. Inaddition, a need remains in the art for α-Gal A preparations with anincreased circulating half-life and increased uptake in specific tissuesother than liver.

SUMMARY OF THE INVENTION

The invention provides highly purified α-Gal A preparations, and variousmethods for purifying the α-Gal A glycoforms. The invention alsoprovides α-Gal A preparations with altered charge and methods for makingthose preparations. Charge alterations are achieved by increasing thesialic acid content of α-Gal A and/or by increasing the phosphorylationof α-Gal A. The invention further provides α-Gal A preparations thathave an extended circulating half-life in a mammalian host, and methodsfor making same. Finally, the present invention further provides methodsand dosages for administering an α-Gal A preparation to a subject. Theα-Gal A preparations of the present invention will be useful fortreatment of individuals with Fabry disease or atypical variants ofFabry disease, e.g., specific populations of Fabry patients withpredominantly cardiovascular abnormalities, such as ventricularenlargement, e.g., left ventricular hypertrophy (LVH), and/or mitralvalve insufficiency, or Fabry patients with predominantly renalinvolvement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the 210 bp probe that was used to isolatean α-Gal A cDNA from a human fibroblast cDNA library (SEQ ID NO:1). Thesequence is from exon 7 of the α-Gal A gene. The probe was isolated fromhuman genomic DNA by the polymerase chain reaction (PCR). The regionsunderlined in the figure correspond to the sequences of theamplification primers.

FIG. 2 is a representation of the sequence of the DNA fragment thatcompletes the 5′ end of the α-Gal A cDNA clone (SEQ ID NO:2). Thisfragment was amplified from human genomic DNA by PCR. The regionsunderlined correspond to the sequences of the amplification primers. Thepositions of the NcoI and SacII restriction endonuclease sites, whichwere used for subcloning as described in Example 1, are also shown.

FIG. 3 is a representation of the sequence of α-Gal A cDNA, includingthe sequence that encodes the signal peptide (SEQ ID NO:3).

FIG. 4 is a schematic map of pXAG-16, an α-Gal A expression constructthat includes the CMV (cytomegalovirus) promoter, exon 1, and firstintron, the hGH signal peptide coding sequence and first intron, thecDNA for α-Gal A (lacking the α-Gal A signal peptide sequence) and thehGH 3′ UTS. pcDNeo indicates the position of the neo gene derived fromplasmid pcDNeo.

FIG. 5 is a schematic map of pXAG-28, an α-Gal A expression constructthat includes the collagen Iα2 promoter and first exon, a β-actinintron, the hGH signal peptide coding sequence and first intron, thecDNA for α-Gal A (lacking the α-Gal A signal peptide sequence) and thehGH 3′ UTS. pcDNco indicates the position of the neo gene derived fromplasmid pcDNeo.

FIG. 6 is a representation of the human α-Gal A amino acid sequence (SEQID NO:4).

FIG. 7 is a representation of the cDNA sequence encoding human α-Gal A(without signal peptide) (SEQ ID NO:5).

FIG. 8 is achromatogram of the α-Gal A purification step using ButylSepharose® resin. The absorbance at 280 nm (plain line) and α-Gal Aactivity (dotted line) of selected fractions is shown.

FIG. 9 is a schematic map of pGA213C.

FIG. 10 is a diagrammatic representation of the targeting construct,pGA213C, and homologous recombination with the endogenousα-galactosidase A locus. pGA213C is depicted as targeting sequencesaligned above corresponding sequences on the X-chromosomalα-galactosidase A locus. Positions relative to the methionine initiationcodon, ATG, are indicated by the numbers above the linear maps. Theactivation unit containing murine dhfr, bacterial neo, and CMVpromoter/aldolase intron sequences is shown above the position (−221)into which they were inserted by DNA cloning. α-galactosidase A codingsequences are indicated by the darkened boxes. α-galactosidase Anon-coding genomic sequences are indicated by the lightly filled boxes.Large arrowheads indicate the direction of transcription for dhfr andneo expression cassettes. Splicing of the GA-GAL mRNA followingsuccessful targeting and gene activation is indicated by the segmentedline below the map of the activated α-galactosidase A (GA-GAL) locus.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The invention described herein relates to certain novel α-Gal Apreparations and methods for making them, as well as methods fortreating patients with Fabry disease or atypical variants of Fabrydisease using those preparations. Certain contemplated representativeembodiments are summarized and described in greater detail below.

The invention uses α-Gal A produced in any cell (an α-Gal A productioncell) for the treatment of Fabry disease. In a preferred embodiment, theinvention uses human α-Gal A produced using standard genetic engineeringtechniques (based on introduction of the cloned α-Gal A gene or cDNAinto a host cell), or gene activation.

The invention provides preparations, and methods for making same, thatcontain a higher purity α-Gal A than prepared in the prior art. Usingthe purification methods of the present invention, compositions of humanα-Gal A preparations are preferably purified to at least 98%homogeneity, more preferably to at least 99% homogeneity, and mostpreferably to at least 99.5% homogeneity, as measured by SDS-PAGE orreverse phase HPLC. The specific activity of the α-Gal A preparations ofthe present invention is preferably at least 2.0×10⁶ units/mg protein,more preferably at least 3.0×10⁶ units/mg protein, and most preferablyat least 3.5×10⁶ units/mg protein.

In one embodiment, α-Gal A preparation is purified by separating thevarious glycoforms of α-Gal A from other components on a hydrophobicinteraction resin, but does not include a lectin chromatography step. Ina preferred embodiment, the functional moiety of the hydrophobicinteraction resin includes a butyl group.

In an alternative embodiment, α-Gal A preparation is purified by firstbinding the various glycoforms of α-Gal A to a cation exchange resin ina column at acidic pH in an equilibration buffer. The column is thenwashed with the equilibration buffer to elute the unbound material, andthe various glycoforms of α-Gal A are eluted using, as an elutionsolution, a salt solution of 10-100 mM, a buffered solution of pH 4-5,or a combination thereof. In a preferred embodiment, the equilibrationbuffer has a pH of about 4.4.

In another alternative embodiment, α-Gal A preparation is purified byseparating the various glycoforms of α-Gal A in a sample from the othercomponents in the sample using a purification procedure comprising astep of at least one of chromatofocusing chromatography, metal chelateaffinity chromatography, or immunoaffinity chromatography as apurification procedure.

The invention further provides α-Gal A preparations and methods formaking α-Gal A preparations that have α-Gal A with altered charge. Thepreparations may include different glycoforms of α-Gal A. Chargealterations are achieved by increasing the sialic acid content of α-GalA preparations and/or by increasing the phosphorylation of α-Gal Apreparations.

The sialic acid content of α-Gal A preparations is increased by (i)isolation of the highly charged and/or higher molecular weight α-Gal Aglycoforms during or after the purification process; (ii) adding sialicacid residues using cells genetically modified (either by conventionalgenetic engineering methods or gene activation) to express a sialyltransferase gene or cDNA; or (iii) fermentation or growth of cellsexpressing the enzyme in a low ammonium environment.

The phosphorylation of α-Gal A preparations is increased by (i) addingphosphate residues using cells genetically modified (either byconventional genetic engineering methods or gene activation) to expressa phosphoryl transferase gene or cDNA; or (ii) adding phosphataseinhibitors to the cultured cells.

Using the methods of the present invention, human glycosylated α-Gal Apreparations are obtained, wherein between 35% and 85% of theoligosaccharides are charged. In a preferred embodiment, at least 35% ofthe oligosaccharides are charged. In a more preferred embodiment, atleast 50% of the oligosaccharides are charged.

Alternative preferred human glycosylated α-Gal A preparations havemultiple α-Gal A glycoforms with preferably at least 20%, morepreferably at least 50%, and most preferably at least 70% complexglycans with 2-4 sialic acid residues. In an alternative preferredembodiment, human glycosylated α-Gal A preparations with multipleglycoforms have an oligosaccharide charge, as measured by the Z number,greater than 100, preferably greater than 150, and more preferablygreater than 170. In another alternative preferred embodiment, humanglycosylated α-Gal A preparations with multiple glycoforms have at leaston average between 16-50%, preferably 25-50%, more preferably at least30%, of glycoforms being phosphorylated. In another alternativeembodiment, the preparations with multiple glycoforms have between50-75%, preferably 60%, of the total glycans being sialylated.

In one embodiment of the present invention, a glycosylated α-Gal Apreparation having an increased oligosaccharide charge is produced byfirst introducing a polynucleotide, which encodes for GlcNAc transferaseIII (GnT-III), into an α-Gal A production cell, or introducing aregulatory sequence by homologous recombination that regulatesexpression of an endogenous GnT-III gene. The α-Gal A production cell isthen cultured under culture conditions which results in expression ofα-Gal A and GnT-III. The final step consists of isolating the α-Gal Apreparation with increased oligosaccharide charge.

In an alternative embodiment of the present invention, a glycosylatedα-Gal A preparation having an increased oligosaccharide charge isproduced by first introducing a polynucleotide, which encodes for asialyl transferase, into an α-Gal A production cell, or introducing aregulatory sequence by homologous recombination that regulatesexpression of an endogenous sialyl transferase gene. The α-Gal Aproduction cell is then cultured under culture conditions which resultsin expression of α-Gal A and the sialyl transferase. The final stepconsists of isolating the α-Gal A preparation with increasedoligosaccharide charge. Preferred sialyl transferases include anα2,3-sialyl transferase and an α2,6-sialyl transferase. In a preferredembodiment, this method includes the additional step of selecting forα-Gal A glycoforms with increased size or increase charge byfractionation or purification of the preparation.

In another embodiment, a glycosylated α-Gal A preparation with increasedsialylation is obtained by contacting an α-Gal A production cell with aculture medium having an ammonium concentration below 10 mM, morepreferably below 2 mM. In a preferred embodiment, the low ammoniumenvironment is achieved by addition of glutamine synthetase to theculture medium. In an alternative preferred embodiment, the low ammoniumenvironment is achieved by continuous or intermittent perfusion of theα-Gal A production cell with fresh culture medium to maintain theammonium concentration below 10 mM, more preferably below 2 mM.

In yet another embodiment, a glycosylated α-Gal A preparation withincreased phosphorylation is obtained by first introducing into an α-GalA production cell a polynucleotide which encodes for phosphoryltransferase, or by introducing a regulatory sequence by homologousrecombination that regulates expression of an endogenous phosphoryltransferase gene. The α-Gal A production cell is then cultured underculture conditions which results in expression of α-Gal A and phosphoryltransferase. The α-Gal A preparation with increased phosphorylationcompared to the α-Gal A produced in a cell without the polynucleotide isthen isolated. In a preferred embodiment, the α-Gal A preparationsproduced by the methods of the present invention have multipleglycoforms with between 16-50%, preferably 25-50%, more preferably atleast 30%, of glycoforms being phosphorylated. In a preferredembodiment, this method includes the additional step of selecting forα-Gal A glycoforms with increased size or increase charge byfractionation or purification of the preparation.

In still another embodiment, a glycosylated α-Gal A preparation withincreased phosphorylation is obtained by adding a phosphatase inhibitor,e.g., bromotetramisole, to cultured cells. Low levels of bovine plasmaalkaline phosphatase can be present in the fetal calf serum used as agrowth additive for cultured cells. This raises the possibility thatexposed Man-6-P epitopes on secreted α-Gal A could be a substrate forserum alkaline phosphatase. Bromotetramisole has been shown to be apotent inhibitor of alkaline phosphatase; Ki=2.8 mM (Metaye et al.,Biochem. Pharmacol. 15: 4263-4268 (1988)) and complete inhibition isachieved at a concentration of 0.1 mM (Borgers & Thone, Histochemistry44: 277-280 (1975)). Therefore, a phosphatase inhibitor, e.g.,bromotetramisole can be added to cultured cells in one embodiment tomaximize the high-uptake form of α-Gal A present in the culture mediumby preventing hydrolysis of the Man-6-P ester groups.

The invention further provides α-Gal A preparations, and methods formaking same, that have an extended circulating half-life in a mammalianhost. The circulating half-life and cellular uptake is enhanced by (i)increasing the sialic acid content of α-Gal A (achieved as above); (ii)increasing the phosphorylation of α-Gal A (achieved as above); (iii)PEGylation of α-Gal A; or (iv) sequential removal of the sialic acid andterminal galactose residues, or removal of terminal galactose residues,on the oligosaccharide chains on α-Gal A.

Improved sialylation of α-Gal A preparations enhances the circulatoryhalf-life of exogenous α-Gal A. In addition, improved sialylation ofα-Gal A improves its uptake, relative to that of hepatocytes, innon-hepatocytes such as liver endothelial cells, liver sinusoidal cells,pulmonary cells, renal cells, neural cells, endothelial cells, orcardiac cells. The human glycosylated α-Gal A preparation with increasedsialic acid content preferably includes multiple glycoforms, with atleast 20% complex glycans having 2-4 sialic acid residues. Analternative preferred human glycosylated α-Gal A preparation hasmultiple glycoforms, wherein between 50-75%, preferably at least 60%, ofthe total glycans are sialylated.

Phosphorylation of α-Gal A preparations also improves the level of α-GalA entering cells. The phosphorylation occurs within the cells expressingthe α-Gal A. One preferred human glycosylated α-Gal A preparation of thepresent invention preferably includes multiple glycoforms with at leaston average between 16-50%, preferably 25-50%, more preferably at least30%, of the glycoforms, being phosphorylated.

In an alternate embodiment, the circulatory half-life of a human α-Gal Apreparation is enhanced by complexing α-Gal A with polyethylene glycol.In a preferred embodiment, the α-Gal A preparation is complexed usingtresyl monomethoxy PEG (TMPEG) to form a PEGylated-α-Gal A. ThePEGylated-α-Gal A is then purified to provide an isolated,PEGylated-α-Gal A preparation. PEGylation of α-Gal A increases thecirculating half-life and in vivo efficacy of the protein.

Sialylation affects the circulatory half-life and biodistribution ofproteins. Proteins with minimal or no sialic acid are readilyinternalized by the asialoglycoprotein receptor (Ashwell receptor) onhepatocytes by exposed galactose residues on the protein. Thecirculating half-life of galactose-terminated α-Gal A can be enhanced bysequentially (1) removing sialic acid by contacting α-Gal A withneuraminidase (sialidase), thereby leaving the terminal galactosemoieties exposed, and (2) removing the terminal galactoside residues bycontacting the desialylated α-Gal A with β-galactosidase. The resultingα-Gal A preparation has a reduced number of terminal sialic acid and/orterminal galactoside residues on the oligosaccharide chains compared toα-Gal A preparations not sequentially contacted with neuraminidase andβ-galactosidase. Alternatively, the circulating half-life ofgalactose-terminated α-Gal A can be enhanced by only removing theterminal galactoside residues by contacting the desialylated α-Gal Awith β-galactosidase. The resulting α-Gal A preparation has a reducednumber of terminal galactoside residues on the oligosaccharide chainscompared to α-Gal A preparations not contacted with β-galactosidase. Ina preferred embodiment, following sequential contact with neuraminidaseand β-galactosidase, the resulting α-Gal A preparations are subsequentlycontacted with β-hexosaminidase, thereby cleaving the oligosaccharide tothe trimannose core.

In addition, sialylation levels can vary depending on the cell typeused. Therefore, in another preferred embodiment, sialylation of α-Gal Acan be enhanced by screening for mammalian cells, e.g., human cells,that have relatively high sialyl transferase activity and using suchcells as α-Gal A production cells.

The invention further provides formulations of an α-Gal A preparationthat are substantially free of non-α-Gal A proteins, such as albumin,non-α-Gal A proteins produced by the host cell, or proteins isolatedfrom animal tissue or fluid. In one embodiment, the formulation furthercomprises an excipient. Preferred excipients include mannitol, sorbitol,glycerol, amino acids, lipids, EDTA, EGTA, sodium chloride, polyethyleneglycol, polyvinylpyrollidone, dextran, or combinations of any of theseexcipients. In another embodiment, the formulation further comprises anon-ionic detergent. Preferred non-ionic detergents include Polysorbate20, Polysorbate 80, Triton X-100, Triton X-114, Nonidet P-40, Octyla-glucoside, Octyl b-glucoside, Brij 35, Pluronic, and Tween 20. In apreferred embodiment, the non-ionic detergent comprises Polysorbate 20or Polysorbate 80. A preferred formulation further comprisesphosphate-buffered saline, preferably at pH 6.

The present invention further provides methods for administering anα-Gal A preparation to a subject. In a preferred embodiment, the α-Gal Apreparation is an α-Gal A preparation with altered charge, e.g.,increased oligosaccharide charge, and/or extended circulating half-lifeas described herein. The dose of administration is preferably between0.05-5.0 mg, more preferably between 0.1-0.3 mg, of the α-Gal Apreparation per kilogram body weight weekly or biweekly. In a preferredembodiment, the dose of administration is about 0.2 mg per kilogram bodyweight biweekly. In these methods, the dose can be administeredintramuscularly, orally, rectally, subcutaneously, intra-arterially,intraperitoneally, intracerebrally, intranasally, intradermally,intrathecally, transmucosally, transdermally, or via inhalation. In oneembodiment, the method for delivering α-Gal A preparation to a subjectcomprises subcutaneously administering a dose ranging between 0.01-10.0mg, preferably 0.1-5.0 mg, of the α-Gal A preparation per kg body weightbiweekly or weekly. The α-Gal A preparation can also be administeredintravenously, e.g., in a intravenous bolus injection, in a slow pushintravenous injection, or by continuous intravenous injection. In any ofthe above methods, the α-Gal A preparation can be delivered using adelivery system such as pump delivery, encapsulated cell delivery,liposomal delivery, needle-delivered injection, needle-less injection,nebulizer, aeorosolizer, electroporation, and transdermal patch. Any ofthe α-Gal A preparation described above can be administered by thesemethods.

An individual who is suspected of having, or known to have, Fabrydisease may be treated by administration of the α-Gal A preparationdescribed above, using the above-described methods of administration anddoses. The present invention contemplates treatment of individuals withFabry disease generally (“Fabry patients”), as well as atypical variantsof Fabry disease, e.g., specific populations of Fabry patients withpredominantly cardiovascular abnormalities, defined here as Fabrypatients with ventricular enlargement, e.g., left ventricularhypertrophy (LVH), and/or mitral valve insufficiency, or Fabry patientswith predominantly renal involvement.

α-Gal A

α-Gal A is a homodimeric glycoprotein that hydrolyses the terminalα-galactosyl moieties from glycolipids and glycoproteins.

The terms mature “α-Gal A” and “GA-GAL” and “SEQ ID NO:5” (see FIG. 7)refer to α-Gal A without a signal peptide (for α-Gal A with the signalpeptide, see FIG. 3 and SEQ ID NO:3). The term “α-Gal A preparation,” asdefined herein, is used interchangeably with the term “glycosylatedα-Gal A preparation” and comprises various glycosylated α-Gal Aglycoforms.

A “signal peptide” is a peptide sequence that directs a newlysynthesized polypeptide to which the signal peptide is attached to theendoplasmic reticulum (ER) for further post-translational processing anddistribution.

An “heterologous signal peptide,” as used herein in the context of α-GalA, means a signal peptide that is not the human α-Gal A signal peptide,typically the signal peptide of some mammalian protein other than α-GalA.

Skilled artisans will recognize that the human α-Gal A DNA sequence(either cDNA [SEQ ID NO:5] or genomic DNA), or sequences that differfrom human α-Gal A DNA due to either silent codon changes or to codonchanges that produce conservative amino acid substitutions, can be usedto genetically modify cultured human cells so that they will overexpressand secrete the enzyme. Certain mutations in the α-Gal A DNA sequencemay encode polypeptides that retain or exhibit improved α-Gal Aenzymatic activity. For example, one would expect conservative aminoacid substitutions to have little or no effect on the biologicalactivity, particularly if they represent less than 10% of the totalnumber of residues in the protein. Conservative substitutions typicallyinclude substitutions within the following groups: glycine, alanine;valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. See, for example, U.S. Pat. No. 5,356,804, incorporated hereinby reference.

Fabry Disease

Fabry disease is a genetic disorder caused by deficient activity of theenzyme α-Gal A. By “α-Gal A deficiency,” it is meant any deficiency inthe amount or activity of this enzyme in a patient, resulting inabnormal accumulations of neutral glycolipids (e.g.,globotriaosylceramide) in histiocytes in blood vessel walls, withangiokeratomas on the thighs, buttocks, and genitalia, hypohidrosis,paresthesia in extremities, cornea verticillata, and spoke-likeposterior subcapsular cataracts. The deposits of this material canresult in pain, serious renal and cardiovascular disease, and stroke.The glycolipid accumulation may induce severe symptoms as typicallyobserved in males who are suffering from Fabry disease. Alternatively,the accumulation may induce relatively mild symptoms, as can sometimesbe seen in heterozygous female carriers of the defective gene. Affectedindividuals have a greatly shortened life expectancy; death usuallyresults from renal, cardiac, or cerebrovascular complications atapproximately age 40. There are no specific treatments for this disease.Fabry disease, classified as a lysosomal storage disorder, affects morethan 15,000 people world-wide.

Fabry disease as defined above is a complex clinical syndromecharacterized by multiorgan and multisystem involvement. Patients whomanifest the combination of corneal dystrophy, skin lesions(angiokeratomata), painful neuropathy, cerebral vascular disease,cardiomyopathy, and renal dysfunction are categorized as displaying the“classic” phenotype. There are, however, patients who manifest some, butnot all aspects of the classic phenotype. These patients are classifiedas “atypical variants of Fabry disease.” There are several atypicalvariant phenotypes associated with α-galactosidase A deficiency. Forexample, some patients with α-galactosidase A deficiency have avariation of Fabry disease with only cardiac involvement, e.g., leftventricular hypertrophy (LVH). There is also another variant phenotypein which patients present with only renal involvement. Although both ofthese variant phenotypes have been defined in male hemizygotes, thevariant forms of Fabry disease have also been described in femaleheterozygotes as well.

Patients with the atypical cardiac variant generally present withsymptomatic disease later in life. The median age of diagnosis forpatients with the cardiac variant phenotype is approximately 52 yearscompared to approximately 29 years for the classic phenotype (Desnick,et al., In The Metabolic and Molecular Bases of Inherited Disease, 6thedition (1996). Scriver, et (eds), McGraw-Hill (New York). pp.2741-2784; Meikle, et al., J. Am. Med. Assoc. 281: 249-254 (1999)).Patients with this syndrome often present with subtle symptoms ofcardiac dysfunction such as exertional dyspnea. Usually, standardechocardiographic analysis reveals that patients with the cardiacvariant phenotype are discovered to have left ventricular hypertrophy(LVH) or asymmetric septal hypertrophy. However, patients may alsopresent with myocardial infarction or cardiomyopathy (Scheidt, et al.,New Engl. J. Med. 324: 395-399 (1991); Nakao, et al., New Engl. J. Med.333: 288-293 (1995)). These patients often undergo myocardial biopsies,and the pathology of the variant syndrome is essentially similar toclassic Fabry disease: myocardial infiltration by deposited glycolipid.α-galactosidase A enzyme assays in these patients reveal a broad rangeof enzyme levels. For example, cardiac variant patients have beenreported to have as high as 30% of the normal levels of α-galactosidaseA enzyme activity, and, thus, up to now have not been considered ascandidates for α-Gal A replacement therapy.

The inventors have now unexpectedly discovered that, although atypicalcardiac variant or atypical renal variant patients may haveα-galactosidase A enzyme activity levels which are relatively highcompared to patients with the classic phenotype of Fabry disease, thesepatients can also benefit from α-galactosidase A enzyme therapy. Forexample, patients can have a mutation which produces a kineticallyunstable α-Gal A enzyme in the cell, and in these patients α-Gal Aenzyme levels can be augmented significantly by administration of α-GalA preparations of the present invention. Also, some patients with theatypical cardiac variant phenotype have been reported to have a pointmutation in amino acid 215 of α-galactosidase A. This amino acid in theunmutated protein is an asparagine which is glycosylated (Eng, et al.,Am. J. Hum. Genet. 53: 1186-1197. (1993)). Thus, α-Gal A enzymereplacement therapy with a properly glycosylated α-galactosidase Apreparations of the present invention can be efficacious in thesepatients. Furthermore, patients with atypical renal variant have beenreported whose only clinical manifestation of Fabry disease is mildproteinuria. Renal biopsy, however, reveals the typical glycolipidinclusions of Fabry disease and α-Gal A enzyme assay reveals lower thannormal levels of α-Gal A. However, because deposited ceramidetrihexoside in the kidney may be detected in shed renal tubular cells inthe urine sediment of these patients, administration of α-Gal Apreparations of the present invention can reduce these levelssubstantially. Lysosomal enzymes such as α-Gal A are targeted to thelysosomal compartment of a cell through interaction with themannose-6-phosphate (M6P) receptor, which binds to M6P residues presentin the oligosaccharide moieties of enzymes destined for the lysosomalcompartment. Kornfeld & Mellman, Ann. Rev. Cell Biol. 5: 483-525 (1989).The primary interaction occurs in the Golgi, where enzymes bound toGolgi M6P receptors are segregated for transport to the lysosomes. Asecondary type of interaction is believed to take place betweenextracellular α-Gal A and M6P receptors at the cell surface. Enzymesthat escape the routing system are secreted by the cell via theconstitutive secretory pathway and are often recaptured by cell surfaceM6P receptors that return the α-galactosidase A to the lysosome by theendocytic pathway. Extracellular substances internalized by cells aretransported through the cytoplasm in endocytic vesicles, which fuse withprimary lysosomes and empty their contents into the lysosomes. In thisprocess, cell surface M6P receptors are also incorporated into endocyticvesicles and transported to lysosomes. In particular, the α-Gal Apreparations of the present invention, in which high levels ofsialylation and/or phosphorylation are present, are preferred for thetreatment of patients with atypical variants of Fabry disease. Suchpreparations, for example, minimize the fraction of the injected α-Gal Athat is removed by hepatocytes and allow high levels of α-Gal A uptakeby non-liver cells, such as renal cells, vascular cells, tubular cells,glomerular cells, cardiac myocytes and cardiac vascular cells.

Extracellular α-Gal A bearing M6P residues may bind to cell surface M6Preceptors and be transported into the lysosomal compartment. Once in thelysosomal compartment, α-Gal A can carry out the appropriate function.It is this aspect of lysosomal enzyme trafficking that makesα-galactosidase A enzyme replacement therapy a feasible therapeutictreatment for Fabry disease patients. Thus, even if a cell isgenetically deficient in producing α-Gal A, the cell may take upextracellular α-Gal A if the α-Gal A is suitably glycosylated and thedeficient cell bears M6P receptors. In patents with Fabry disease,vascular endothelial cells of the kidney and heart display severehistopathologic abnormalities and contribute to the clinical pathologyof the disease. These cells, which carry M6P receptors, are a particulartherapeutic target of α-Gal A. An object of the invention is to providean α-Gal A preparation in which M6P is present in the N-linkedoligosaccharides.

The degree to which the N-linked oligosaccharides of α-Gal A aremodified by sialylation has a substantial effect on α-Gal Apharmacokinetics and biodistribution. In the absence of appropriatesialylation, α-Gal A is rapidly cleared from the circulation due tobinding by hepatic asialoglycoprotein receptors (Ashwell receptors),followed by internalization and degradation by hepatocytes. Ashwell &Harford, Ann. Rev. Biochem. 51: 531-554 (1982). This decreases theamount of α-Gal A available in the circulation for binding to M6Preceptors on cells which contribute to the clinical pathology of Fabrydisease, such as the vascular endothelial cells of the kidney and heart.α-Gal A secreted by genetically-modified human cells has glycosylationproperties which are suitable for the treatment of Fabry disease byeither conventional pharmaceutical administration of the purifiedsecreted protein or by gene therapy, without requiring additionalenzymatic modification as has been reported to be required for thelysosomal enzyme, glucocerebrosidase, in which uptake of purifiedglucocerebrosidase enzyme by clinically-relevant cells requires complexenzymatic modification of the enzyme following purification from humanplacenta. Beutler, New Engl. J. Med. 325: 1354-1360 (1991).

Cells Suitable for Production of α-Gal A

An individual suspected of having an α-Gal A deficiency such as Fabrydisease can be treated with purified human α-Gal A obtained fromcultured, genetically-modified cells, preferably human cells.

When cells are to be genetically modified for the purposes of treatmentof Fabry disease, the cells may be modified by conventional geneticengineering methods or by gene activation.

According to conventional methods, a DNA molecule that contains an α-GalA cDNA or genomic DNA sequence may be contained within an expressionconstruct and transfected into primary, secondary, or immortalized cellsby standard methods including, but not limited to, liposome-,polybrene-, or DEAE dextran-mediated transfection, electroporation,calcium phosphate precipitation, microinjection, or velocity drivenmicroprojectiles (“biolistics”) (see, e.g., a copending application,U.S. Ser. No. 08/334,797, incorporated herein by reference).Alternatively, one could use a system that delivers the geneticinformation by viral vector. Viruses known to be useful for genetransfer include adenoviruses, adeno-associated virus, herpes virus,mumps virus, poliovirus, retroviruses, Sindbis virus, and vaccinia virussuch as canary pox virus.

Alternatively, the cells may be modified using a gcnc activation (“GA”)approach, such as described in U.S. Pat. Nos. 5,733,761 and 5,750,376,each incorporated herein by reference. α-Gal A made by gene activationis referred to herein as GA-GAL.

Accordingly, the term “genetically modified,” as used herein inreference to cells, is meant to encompass cells that express aparticular gene product following introduction of a DNA moleculeencoding the gene product and/or regulatory elements that controlexpression of a coding sequence for the gene product. The DNA moleculemay be introduced by gene targeting or homologous recombination, i.e.,introduction of the DNA molecule at a particular genomic site.Homologous recombination may be used to replace the defective geneitself (the defective α-Gal A gene or a portion of it could be replacedin a Fabry disease patient's own cells with the whole gene or a portionthereof).

As used herein, the term “primary cell” includes cells present in asuspension of cells isolated from a vertebrate tissue source (prior totheir being plated, i.e., attached to a tissue culture substrate such asa dish or flask), cells present in an explant derived from tissue, bothof the previous types of cells plated for the first time, and cellsuspensions derived from these plated cells.

“Secondary cells” refers to cells at all subsequent steps in culturing.That is, the first time a plated primary cell is removed from theculture substrate and replated (passaged), it is referred to as asecondary cell, as are all cells in subsequent passages.

A “cell strain” consists of secondary cells which have been passaged oneor more times; exhibit a finite number of mean population doublings inculture; exhibit the properties of contact-inhibited, anchoragedependent growth (except for cells propagated in suspension culture);and are not immortalized.

By “immortalized cell” is meant a cell from an established cell linethat exhibits an apparently unlimited lifespan in culture.

Examples of primary or secondary cells include fibroblasts, epithelialcells including mammary and intestinal epithelial cells, endothelialcells, formed elements of the blood including lymphocytes and bonemarrow cells, glial cells, hepatocytes, keratinocytes, muscle cells,neural cells, or the precursors of these cell types. Examples ofimmortalized human cell lines useful in the present methods include, butare not limited to, Bowes Melanoma cells (ATCC Accession No. CRL 9607),Daudi cells (ATCC Accession No. CCL 213), HeLa cells and derivatives ofHeLa cells (ATCC Accession Nos. CCL 2, CCL 2.1, and CCL 2.2), HL-60cells (ATCC Accession No. CCL 240), HT-1080 cells (ATCC Accession No.CCL 121), Jurkat cells (ATCC Accession No. TIB 152), KB carcinoma cells(ATCC Accession No. CCL 17), K-562 leukemia cells (ATCC Accession No.CCL 243), MCF-7 breast cancer cells (ATCC Accession No. BTH 22), MOLT-4cells (ATCC Accession No. 1582), Namalwa cells (ATCC Accession No. CRL1432), Raji cells (ATCC Accession No. CCL 86), RPMI 8226 cells (ATCCAccession No. CCL 155), U-937 cells (ATCC Accession No. CRL 1593),WI-38VA13 sub line 2R4 cells (ATCC Accession No. CLL 75.1), CCRF-CEMcells (ATCC Accession No. CCL 119), and 2780AD ovarian carcinoma cells(Van der Blick et al., Cancer Res. 48: 5927-5932, 1988), as well asheterohybridoma cells produced by fusion of human cells and cells ofanother species.

Following the genetic modification of human cells to produce a cellwhich secretes α-Gal A, a clonal cell strain consisting essentially of aplurality of genetically identical cultured primary human cells or,where the cells are immortalized, a clonal cell line consistingessentially of a plurality of genetically identical immortalized humancells, may be generated. In one embodiment, the cells of the clonal cellstrain or clonal cell line are fibroblasts. In a preferred embodimentthe cells are secondary human fibroblasts, e.g., BRS-11 cells.

After genetic modification, the cells are cultured under conditionspermitting secretion of α-Gal A. The protein is isolated from thecultured cells by collecting the medium in which the cells are grown,and/or lysing the cells to release their contents, and then applyingprotein purification techniques.

Purification of α-Gal A from the Conditioned Medium of StablyTransfected Cells

According to the methods of this invention, the α-Gal A protein isisolated from the cultured cells (“α-Gal A production cells”) bycollecting the medium in which the cells are grown, or lysing the cellsto release their contents, and then applying protein purificationtechniques without the use of lectin affinity chromatography. Thepreferred purification process is outlined in Example 2 below.

Alternative hydrophobic interaction resins, such as Source Iso(Pharmacia), Macro-Prep® Methyl Support (Bio-Rad), TSK Butyl (Tosohaas)or Phenyl Sepharose® (Pharmacia), can also be used to purify α-Gal A.The column can be equilibrated in a relatively high concentration of asalt, e.g., −1 M ammonium sulfate or 2 M sodium chloride, in a buffer ofpH 5.6. The sample to be purified is prepared by adjusting the pH andsalt concentration to those of the equilibration buffer. The sample isapplied to the column and the column is washed with equilibration bufferto remove unbound material. The α-Gal A is eluted from the column with alower ionic strength buffer, water, or organic solvent in water, e.g.,20% ethanol or 50% propylene glycol. Alternatively, the α-Gal A can bemade to flow through the column by using a lower concentration of saltin the equilibration buffer and in the sample or by using a differentpH. Other proteins may bind to the column, resulting in purification ofthe α-Gal A-containing sample which did not bind the column. A preferredfirst purification step is the use of a hydroxyapatite column.

An alternative step of purification can use a cation exchange resin,e.g., SP Sepharose® 6 Fast Flow (Pharmacia), Source 30S (Pharmacia), CMSepharose® Fast Flow (Pharmacia), Macro-Prep® CM Support (Bio-Rad) orMacro-Prep® High S Support (Bio-Rad), to purify α-Gal A. The “firstchromatography step” is the first application of a sample to achromatography column (all steps associated with the preparation of thesample are excluded). The α-Gal A can bind to the column at pH 4.4. Abuffer, such as 10 mM sodium acetate, pH 4.4, 10 mM sodium citrate, pH4.4, or other buffer with adequate buffering capacity at approximatelypH 4.4, can be used to equilibrate the column. The sample to be purifiedis adjusted to the pH and ionic strength of the equilibration buffer.The sample is applied to the column and the column is washed after theload to remove unbound material. A salt, such as sodium chloride orpotassium chloride, can be used to elute the α-Gal A from the column.Alternatively, the α-Gal A can be eluted from the column with a bufferof higher pH or a combination of higher salt concentration and higherpH. The α-Gal A can also be made to flow through the column duringloading by increasing the salt concentration in the equilibration bufferand in the sample load, by running the column at a higher pH, or by acombination of both increased salt and higher pH.

Another step of purification can use a Q Sephrarose® 6 Fast Flow for thepurification of α-Gal A. Q Sepharose® 6 Fast Flow is a relatively stronganion exchange resin. A weaker anion exchange resin such as DEAESepharose® Fast Flow (Pharmacia) or Macro-Prep® DEAB (Bio-Rad) can alsobe used to purify α-Gal A. The column is equilibrated in a buffer, e.g.,10 mM sodium phosphate, pH 6. The pH of the sample is adjusted to pH 6,and low ionic strength is obtained by dilution or diafiltration of thesample. The sample is applied to the column under conditions that bindα-Gal A. The column is washed with equilibration buffer to removeunbound material. The α-Gal A is eluted with application of salt, e.g.,sodium chloride or potassium chloride, or application of a lower pHbuffer, or a combination of increased salt and lower pH. The α-Gal A canalso be made to flow through the column during loading by increasing thesalt concentration in the load or by running the column at a lower pH,or by a combination of both increased salt and lower pH.

Another step of purification can use a Superdex® 200 (Pharmacia) sizeexclusion chromatography for purification of α-Gal A. Other sizeexclusion chromatography resins such as Sephacryl® S-200 HR or Bio-Gel®A-1.5 m can also be used to purify α-Gal A. The preferred buffer forsize exclusion chromatography is 25 mM sodium phosphate, pH 6.0,containing 0.15 M sodium chloride. Other formulation-compatible bufferscan also be used, e.g., 10 mM sodium or potassium citrate. The pH of thebuffer can be between pH 5 and pH 7 and should at contain a salt, e.g.,sodium chloride or a mixture of sodium chloride and potassium chloride.

Another step of purification can use a chromatofocusing resin such asPolybuffer Exchanger PBE 94 (Pharmacia) to purify α-Gal A. The column isequilibrated at relatively high pH (e.g., pH 7 or above), the pH of thesample to be purified is adjusted to the same pH, and the sample isapplied to the column. Proteins are eluted with a decreasing pH gradientto a pH such as pH 4, using a buffer system, e.g., Polybuffer 74(Pharmacia), which had been adjusted to pH4.

Alternatively, immunoaffinity chromatography can be used to purify α-GalA. An appropriate polyclonal or monoclonal antibody to α-Gal A(generated by immunization with α-Gal A or with a peptide derived fromthe α-Gal A sequence using standard techniques) can be immobilized on anactivated coupling resin, e.g., NHS-activated Sepharose® 4 Fast Flow(Pharmacia) or CNBr-activated Sepharose® 4 Fast Flow (Pharmacia). Thesample to be purified can be applied to the immobilized antibody columnat about pH 6 or pH 7. The column is washed to remove unbound material.α-Gal A is eluted from the column with typical reagents utilized foraffinity column elution such as low pH, e.g., pH 3, denaturant, e.g.,guanidine HCl or thiocyanate, or organic solvent, e.g., 50% propyleneglycol in a pH 6 buffer. The purification procedure can also use a metalchelate affinity resin, e.g., Chelating Sepharose® Fast Flow(Pharmacia), to purify α-Gal A. The column is pre-charged with metalions, e.g., Cu²⁺, Zn²⁺, Ca²⁺, Mg²⁺ or Cd²⁺. The sample to be purified isapplied to the column at an appropriate pH, e.g., pH 6 to 7.5, and thecolumn is washed to remove unbound proteins. The bound proteins areeluted by competitive elution with imidazole or histidine or by loweringthe pH using sodium citrate or sodium acetate to a pH less than 6, or byintroducing chelating agents, such as EDTA or EGTA.

According to the foregoing protocols, this invention providespreparations with a higher purity α-Gal A preparation than prepared inthe prior art, purified to at least 98% homogeneity, more preferably toat least 99% homogeneity, and most preferably to at least 99.5%homogeneity, as measured by SDS-PAGE or reverse phase HPLC. The α-Gal Apreparations of the present invention may comprise numerous α-Gal Aglycoforms. Accordingly, the term “homogeneity,” as used herein in thecontext of α-Gal A preparations, refers to preparations that aresubstantially free (<2% of the total proteins) of proteins other thanα-Gal A. Examples of non-α-Gal A proteins such as albumin, non-α-Gal Aproteins produced by the host cell, and non-α-Gal A proteins isolatedfrom animal tissue or fluid. The specific activity of the α-Gal Apreparations of the present invention is preferably at least 2.0×10⁶units/mg protein, more preferably at least 3.0×10⁶ units/mg protein, andmost preferably at least 3.5×10⁶ units/mg protein.

Improving Circulating Half-Life of α-Gal A Preparations by GlycanRemodeling to Increase Oligosaccharide Charge

The invention provides a glycoprotein modification program for increaseduptake of a therapeutic enzyme in specific tissues other than liver andmacrophages. Using the methods of the present invention, humanglycosylated α-Gal A preparations are obtained, wherein between 35% and85% of the oligosaccharides are charged, preferably at least 50% of theoligosaccharides being charged.

Protein N-glycosylation functions by modifying appropriate asparagineresidues of proteins with oligosaccharide structures, thus influencingtheir properties and bioactivities. Kukuruzinska & Lennon, Crit. Rev.Oral. Biol. Med. 9: 415-48 (1998). The present invention provides anisolated α-Gal A preparation in which a high percentage of theoligosaccharides are negatively charged, primarily by the addition ofone to four sialic acid residues on complex glycans, or of one to twophosphate moieties on high-mannose glycans, or of a single phosphate anda single sialic acid on hybrid glycans. Smaller amounts of sulfatedcomplex glycans may also be present. A high proportion of chargedstructures serves two main functions. First, capping of penultimategalactose residues by 2,3- or 2,6-linked sialic acid prevents prematureremoval from the circulation by the asialoglycoprotein receptor presenton hepatocytes. This receptor recognizes glycoproteins with terminalgalactose residues. Increasing the circulatory half-life of α-Gal Agives important target organs such as heart and kidney the opportunityto endocytose greater amounts of enzyme from the plasma following enzymeinfusion. Second, the presence of Man-6-phosphate on high-mannose orhybrid glycans provides an opportunity for receptor-mediated uptake bythe cation-independent Man-6-phosphate receptor (CI-MPR). Thisreceptor-mediated uptake occurs on the surface of many cells, includingvascular endothelial cells, which are a major storage site of CTH inFabry patients. Enzyme molecules with two Man-6-phosphate residues havea much greater affinity for the CI-MPR than those with a singleMan-6-phosphate. Representative glycan structures are provided in Table1.

TABLE 1 Representative Glycan Structures A biantennary glycan:

A tetraantennary glycan:

A high-mannose glycan:

A phosphorylated hybrid glycan:

A bisphosphorylated glycan:

N-glycoprotein biosynthesis involves a multitude of enzymes,glycosyltransferases, and glycosidases. The majority of these enzymesfunction in the endoplasmic reticulum (ER) and Golgi apparatus in anordered and well-orchestrated manner. The complexity of N-glycosylationis augmented by the fact that different asparagine residues within thesame polypeptide may be modified with different oligosaccharidestructures, and various proteins are distinguished from one another bythe characteristics of their carbohydrate moieties. Recent advances inmolecular genetics have expedited the identification, isolation, andcharacterization of N-glycosylation genes. As a result, informationregarding relationships between N-glycosylation and other cellularfunctions has emerged.

N-linked glycoprotein processing in the cell begins when anoligosaccharide chain with a Glc₃Man₉GlcNAc₂ is added to an acceptorasparagine on a nascent peptide in the lumen of the ER as a single unit.A fourteen sugar oligosaccharide chain consisting of Glc₃Man₉GlcNAc₂ isbuilt up on dolichol, a very long chain aliphatic alcohol:

This oligosaccharide is transferred as a single unit to an acceptorasparagine residue on a nascent peptide chain in the lumen of the ER.The large size of the glycan relative to the peptide may guide proteinfolding. The three glucose residues serve as a signal that theoligosaccharide is completed and ready for transfer by oligosaccharyltransferase. This enzyme will also transfer nonglucosylatedoligosaccharides but at only a fraction of the rate of the completedchain because these are sub-optimal substrates. One form of carbohydratedeficient glycoprotein syndrome in humans has been shown to be caused bya deficiency of Dolichol-P-Glc: Man₉GlcNAc₂-PP-Dolichol glucosyltransferase, the first enzyme in the glucose addition pathway, whichresults in hypoglycosylation of serum proteins. Korner et al., Proc.Natl. Acad. Sci. USA 95: 13200-13205 (1998). After removal of the threeglucose residues and achievement of the correct conformation, the newlysynthesized glycoprotein is exported to the Golgi. Depending on theaccessibility of the glycan to Golgi mannosidases after protein folding,the glycan chain may stay as a high mannose chain with 5-9 mannoseresidues. Alternatively, the glycan chain may be further processed to atrimannosyl core, and become an acceptor for other glycosyl transferasesthat form complex chains by addition of more GlcNAc residues, followedby Gal, NeuAc and Fuc. A third possibility, if the protein has twolysine residues exactly 34 angstroms apart and in the correct spatialrelationship to a high mannose chain, is the addition of GlcNAcα-1-PO₄onto carbon 6 of one, or sometimes two, mannose residues. Cuozzo et al.,J. Biol. Chem. 273: 21069-21076 (1998). After removal of the α-linkedGlcNAc by a specific enzyme, a terminal M6P epitope is generated whichis recognized by a M6P receptor in the trans Golgi network that thentargets these enzymes to lysosomes in cells of mesenchymal origin.

To target α-Gal A to as many different tissues as possible, manydifferent carbohydrate structures (glycoforms) are useful. Matsuura etal., Glycobiology 8: 329-339 (1998) reported that the glycan structureson human α-Gal A made in CHO cells had 41% high-mannose glycans and thephosphorylation level was 24%. However, the level of sialylated complexglycans was only 11%. Thus, ⅔ of the complex chains were not sialylated,which results in the rapid elimination of α-Gal A by the liver. Theα-Gal A produced in the human cells of the invention has a higherpercentage of charged oligosaccharides than the prior art α-Gal Aproduced in CHO cells. For example, α-Gal A synthesized in HT-1080 cellsdescribed herein is particularly suitable, because α-Gal A produced inHT-1080 cells contains approximately 15% neutral structures(high-mannose and hybrid), approximately 16% phosphorylated glycans, andapproximately 67% complex glycans with 2 to 4 sialic acid residues.Thus, essentialy all of the complex chains are sialylated as compared toα-Gal A produced in CHO cells. HT-1080 cell α-Gal A has three N-linkedglycosylation sites. Two sites are processed to complex glycans in theGolgi apparatus, while the third site is occupied by a high-mannoseglycan, 50% of which is modified by lysosomal enzyme-specificphosphorylation to yield both monophosphorylated and diphosphorylatedspecies.

Four approaches are provided for carbohydrate remodeling on a proteincontaining N-linked glycan chains. First, the proportion of chargedα-Gal A can be increased by selective isolation of glycoforms during thepurification process. The present invention provides for increasing theproportion of highly charged and higher molecular weight α-Gal Aglycoforms by fractionation of α-Gal A species on chromatography columnresins during and/or after the purification process. The more highlycharged glycoform species of α-Gal A contain more sialic acid and/ormore phosphate, and the higher molecular weight glycoforms would alsocontain the fully glycosylated, most highly branched and highly chargedspecies. Selection of the charged species, or removal of thenon-glycosylated, poorly glycosylated or poorly sialylated and/orphosphorylated α-Gal A species would result in a population of α-Gal Aglycoforms with more sialic acid and/or more phosphate, thereforeproviding an α-Gal A preparation with higher half-life and potentialtherapeutic efficiency.

This fractionation process can occur on, but is not limited to, suitablechromatographic column resins utilized to purify or isolate α-Gal A. Forexample, fractionation can occur on, but is not limited to, cationexchange resins (such as SP-Sepharose®), anion exchange resins(Q-Sepharose®), affinity resins (Heparin Sepharose®, lectin columns)size exclusion columns (Superdex® 200) and hydrophobic interactioncolumns (Butyl Sepharose®) and other chromatographic column resins knownin the art.

Since α-Gal A is produced in cells as a heterogeneous mixture ofglycoforms which differ in molecular weight and charge, α-Gal A tends toelute in relatively broad peaks from the chromatography resins. Withinthese elutions, the glycoforms are distributed in a particular mannerdepending on the nature of the resin being utilized. For example, onsize exclusion chromatography, the largest glycoforms will tend to eluteearlier on the elution profile than the smaller glycoforms.

On ion exchange chromatography, the most negatively charged glycoformswill tend to bind to a positively charged resin (such as Q-Sepharose®)with higher affinity than the less negatively charged glycoforms, andwill therefore tend to elute later in the elution profile. In contrast,these highly negatively charged glycoforms may bind less tightly to anegatively charged resin, such as SP Sepharose®, than less negativelycharges species, or may not even bind at all.

Fractionation of the glycoform species on chromatographic resins can beinfluenced by pH, ionic strength, buffer salt selection, viscosityand/or other parameters such choice of resin type. The use of varioustypes of gradient elutions (straight line linear gradients, curved,e.g., exponential gradients) or use of a series of short step elutionsto selectively elute α-Gal A species from the chromatography column canalso be optimized for α-Gal A fractionation. All of these factors, aloneor in combination, can be optimized to achieve efficient fractionationof the glycoforms. Fractionation can also occur after the purificationprocess is completed, on a particular chromatographic resin selectivelyoptimized for the fractionation and selection of the desired glycoformpopulation.

Selection of glycoform populations from the fractionated α-Gal A speciescan be achieved after analysis of the eluted α-Gal A glycoforms. Theelution peak can be analyzed by various techniques such as, but notlimited to, SDS-PAGE, isoelectric focusing, capillary electrophoresis,analytical ion exchange HPLC, and/or analytical size exclusion HPLC.Particular fractions can be selected which tend towards the desired sizeor charge profile. Selection can occur at every chromatographic step inthe process, allowing for gradual achievement of the desired glycoformpopulation, or can be limited to a particular step or steps if theefficiency of fractionation of the step(s) is high. Fractionation canalso occur after the purification process is completed, on a particularchromatographic resin selectively optimized for the fractionation andselection of the desired glycoform population.

Fractionation and selection of highly charged and/or higher molecularweight glycoforms of α-Gal A can be performed on any α-Gal Apreparation, such as that derived from genetically modified cells suchas cells modified by conventional genetic engineering methods or by geneactivation (GA). It can be performed on cell lines grown in optimizedsystems to provide higher sialylation and phosphorylation as describedabove, or PEGylated α-Gal A as described below.

For example, in the α-Gal A purification process as described herein,fractionation of α-Gal A glycoforms can occur at various steps in theprocess. On the hydrophobic resin, Butyl Sepharose® Fast Flow, thehighest charged α-Gal A glycoforms elute first, followed by the lesshighly charges species. For Heparin Sepharose®, the highest chargedspecies also elute first in the elution peak, followed by the lesshighly charged species. The opposite occurs with Q-Sepharose®, where theleast highly charged species eluting first, followed by the most highlycharged glycoforms. On size exclusion chromatography on Superdex® 200,the highest molecular weight glycoforms elute first followed by thelower molecular weight, less glycosylated α-Gal A species. To allow forefficient fractionation of particular α-Gal A glycoform populations,multiple chromatographic steps can be combined, all of which fractionateon different physical methods. For example, to obtain the α-Gal Aglycoforms containing the lowest pI (those containing the most negativecharge) limiting the pooling the early eluting butyl fractions wouldenhance for the more highly charged α-Gal A. Proceeding with thisselected pool on the Heparin column, and again limiting the pooling tothe earlier, more highly negatively charged α-Gal A species furtherenhances the proportion of low pI α-Gal A glycoforms in the pool.Further fine tuning of the glycoform population can be done at varioussteps of the purification process by monitoring the size and chargedistribution of the elution pools by SDS-PAGE and isoelectric focusing.An example of fractionation by size and charge is outlined below inExample 2.4.

The second approach for carbohydrate remodeling involves modifyingcertain glycoforms on the purified α-Gal A by attachment of anadditional terminal sugar residue using a purified glycosyl transferaseand the appropriate nucleotide sugar donor. This treatment affects onlythose glycoforms that have an appropriate free terminal sugar residue toact as an acceptor for the glycosyl transferase being used. For example,α2,6-sialyltransferase adds sialic acid in an a 2,6-linkage onto aterminal Galβ1,4GlcNAc-R acceptor, using CMP-sialic acid as thenucleotide sugar donor. Commercially available enzymes and their speciesof origin include: fucose α1,3 transferases III, V and VI (humans);galactose α1,3 transferase (porcine); galactose β1,4 transferase(bovine); mannose α1,2 transferase (yeast); sialic acid α2,3 transferase(rat); and sialic acid α2,6 transferase (rat). After the reaction iscompleted, the glycosyl transferase can be removed from the reactionmixture by a glycosyl transferase specific affinity column consisting ofthe appropriate nucleotide bonded to a gel through a 6 carbon spacer bya pyrophosphate (GDP, UDP) or phosphate (CMP) linkage or by otherchromatographic methods known in the art. Of the glycosyl transferaseslisted above, the sialyl transferases is particularly useful formodification of enzymes, such as α-Gal A, for enzyme replacement therapyin human patients. Use of either sialyl transferase withCMP-5-fluoresceinyl-neuraminic acid as the nucleotide sugar donor yieldsa fluorescently labeled glycoprotein whose uptake and tissuelocalization can be readily monitored.

The third approach for carbohydrate remodeling involvesglyco-engineering, e.g., introduction of genes that affect glycosylationmechanisms of the cell, of the α-Gal A production cell to modifypost-translational processing in the Golgi apparatus is a preferredapproach.

The fourth approach for carbohydrate remodeling involves treating α-GalA with appropriate glycosidases to reduce the number of differentglycoforms present. For example, sequential treatment of complex glycanchains with neuraminidase, β-galactosidase, and β-hexosaminidase cleavesthe oligosaccharide to the trimannose core.

The structure of an N-linked glycan depends on the accessibility of theglycan chain to Golgi processing mannosidases after the protein hasfolded, and the presence in the Golgi of a family of glycosyltransferases and the appropriate nucleotide sugar donors. Many of theglycosyl transferases catalyze competing reactions, which can result inthe glycan chain being elongated in several different and compatibleways, depending on which enzyme reacts first. This results inmicroheterogeneity and the formation of a complex family of glycoforms.Some structures are unique to a single tissue, such as the modificationof certain pituitary hormones by the addition of GalNAc-4-SO₄, or arelimited to a few organs.

An example of the latter is the formation of a so-called bisectingGlcNAc (GlcNAc linked β1,4 to the core β-mannose residue) on complexglycans of glutamyltranspeptidase in kidney, but not in liver. Abisected biantennary structure on γ-glutamyltranspeptidase is shownbelow:

In mammals, the enzyme responsible, GlcNAc transferase III (GnT-III), isfound in certain cells of the brain and kidney and in certain cells ofthe liver in patients with hepatocarcinomas. GnT-III catalyzes theaddition of N-acetylglucosamine in β1-4 linkage to the β-linked mannoseof the trimannosyl core of N-linked sugar chains to produce a bisectingGlcNAc residue. The mouse, rat, and human genes for GnT-III have beencloned. Ihara et al., J. Biochem. (Tokyo) 113: 692-698 (1993).

The presence of additional GlcNAc T-III activity in human cells canproduce an increase in monophosphorylated hybrid glycans at the expenseof bi-, tri-, and tetrantennary complex glycans. This should not affectthe plasma half-life adversely, but may increase targeting to vascularendothelial cells. A representative structure is shown below:

Some of the α-Gal A is taken up by the kidney and results in asignificant decrease in the stored glycolipids. Because the kidney canform N-glycans with bisecting GlcNAc residues, renal epithelial cellscan recognize glycoproteins with this epitope with a particularly highspecificity.

Elevated GnT-III activity can cause an imbalance in branching on thetrimannosyl core by inhibiting further branching by GnT-II, IV, V, andGal β1,4-transferase at the substrate level. Recently, a Chinese hamsterovary (CHO) cell line capable of producing bisected oligosaccharides onglycoproteins was created by overexpression of recombinant GnT-III.Sburlati et al., Biotechnol. Progr. 14: 189-192 (1998). Interferon β(IFN-β) was chosen as a model and potential therapeutic secretedheterologous protein on which the effect of GnT-III-expression onproduct glycosylation could be evaluated. IFN-β with bisectedoligosaccharides was produced by the GnT-III-engineered CHO cells, butnot by the unmodified parental cell line.

The production of glycoprotein therapeutics requires characterization ofglycosylation with respect to the lot-to-lot consistency. The‘hypothetical N-glycan charge Z’ has been used as a parameter tocharacterize the protein glycosylation in a simple, efficient manner.The determination of Z has been validated in multiple repetitiveexperiments and proved to be highly accurate and reliable. Hermentin etal., Glycobiology 6: 217-230 (1996). The hypothetical N-glycan charge ofa given glycoprotein is deduced from the N-glycan mapping profileobtained via high performance anion-exchange chromatography(HPAEC)/pulsed amperometric detection (PAD). In HPAEC, N-glycans areclearly separated according to their charge, e.g., their number ofsialic acid residues, providing distinct regions for neutral structuresas well as for the mono- di-, tri-, and tetrasialylated N-glycans. Z isdefined as the sum of the products of the respective areas (A) in theasialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region,each multiplied by the corresponding charge:

Z=A _((asialo)) ·+A _((MS))·1+A _((DiS))·2+A _((TriS))·3+A_((TetraS))·4[+A _((pentaS))·5]

Z=ΣA _((i))·(i)

where i is 0 in the asialo region, 1 in the monosialo (MS) region, 2 inthe disialo (DiS) region, 3 in the trisialo (TriS) region, 4 in thetetrasialo (TetraS) region, and 5 in the pentasialo (PentaS) region.

Thus, a glycoprotein with mostly C4-4* structures will provide Z≅400, aglycoprotein carrying largely C2-2* structures will amount to Z≅200, anda glycoprotein carrying only high-mannose type or truncated structureswill provide Z≅0.

Human glycosylated α-Gal A preparations of the present invention have anoligosaccharide charge, as measured by the Z number, greater than 100,preferably greater than 150, and more preferably greater than 170.

Altering the Half-Life of Serum α-Gal A by Phosphorylation

Phosphorylation of α-Gal A may be altered to affect the circulatinghalf-life of α-Gal A and the level of α-Gal A entering cells. Thephosphorylation is preferably achieved within the cell expressing α-GalA. Specifically contemplated is obtaining a glycosylated α-Gal Apreparation with increased phosphorylation by first introducing into anα-Gal A producing-cell a DNA sequence which encodes for phosphoryltransferase, or by introducing a regulatory sequence by homologousrecombination that regulates expression of an endogenous phosphoryltransferase gene. The α-Gal A production cell is then cultured underculture conditions which result in expression of α-Gal A and phosphoryltransferase. Isolation can then be performed of the α-Gal A preparationwith increased phosphorylation compared to the α-Gal A produced in acell without the polynucleotide. Such phosphoryl transferases are wellknown in the art. See, for example, U.S. Pat. Nos. 5,804,413 and5,789,247, each incorporated herein by reference.

The concerted actions of two membrane-bound Golgi enzymes are needed togenerate a Man-6-phosphate recognition marker on a lysosomal proenzyme.The first, UDP-N-acetylglucosamine: glycoproteinN-acetylglucosamine-1-phosphotransferase (GlcNAc phosphotransferase),requires a protein recognition determinant on lysosomal enzymes thatconsists of two lysine residues exactly 34 Å apart and in the correctspatial relationship to a high mannose chain. The second,N-acetylglucosamine-1-phosphodiestera-N-acetylglucosaminidase(phosphodiester α-GlcNAcase), hydrolyzes the α-GlcNAc-phosphate bondexposing the Man-6-phosphate recognition site.

According to the methods of this invention, the α-Gal A preparationsproduced by the methods of the present invention have multipleglycoforms with between 16-50%, preferably 25-50%, more preferably atleast 30%, of glycoforms being phosphorylated.

Altering the Half-Life of Serum α-Gal A by Increased Sialylation

Increased sialylation of undersialylated glycans with terminal galactoseresidues can be accomplished by transfection of mammalian and preferablyhuman cells with sialyl transferase gene.

The present invention provides a glycosylated α-Gal A preparation havingan increased oligosaccharide charge produced by first introducing apolynucleotide, which encodes for sialyl transferase, into an α-Gal Aproducing-cell, or introducing a regulatory sequence by homologousrecombination that regulates expression of an endogenous sialyltransferase gene. The α-Gal A production cell is then cultured underculture conditions which result in expression of α-Gal A and sialyltransferase. The following step consists of isolating the α-Gal Apreparation with increased oligosaccharide charge. Preferred sialyltransferases include an α2,3-sialyltransferase and an α2,6-sialyltransferase. These sialyl transferases are well known. For example, seeU.S. Pat. No. 5,858,751, incorporated herein by reference.

In a preferred embodiment, this method of increasing sialylationincludes the additional step of selecting for α-Gal A glycoforms withincreased size or increased charge by fractionation or purification ofthe preparation (as discussed below).

Alternatively, the invention provides for increasing sialylation bymaintaining cells in a low ammonium environment. In particular, aglycosylated α-Gal A preparation with increased sialylation is obtainedby contacting an α-Gal A production cell with a culture medium having anammonium concentration below 10 mM, more preferably below 2 mM.Increased sialylation can be accomplished by perfusion of productioncells by which toxic metabolites, such as ammonia, are periodicallyremoved from the culture medium. In a preferred embodiment, the lowammonium environment is achieved by addition of the glutamine synthetasegene or cDNA to the production cells. Alternatively, the low ammoniumenvironment is achieved by perfusion of the α-Gal A production cell withfresh culture medium to maintain the ammonium concentration below 10 mM,more preferably below 2 mM. The production cells may be perfusedcontinuously with fresh culture medium with an ammonium concentrationbelow 10 mM, more preferably below 2 mM. Alternatively, the productioncells may be perfused intermittently with fresh culture medium.Intermittent perfusion, as used herein, refers to either perfusion atregular, periodic intervals of time, or after a measurement of theammonium concentration approaching the target concentration (i.e., 10mM, more preferably below 2 mM). The intermittent perfusions should beat intervals sufficiently frequent such that the ammonium concentrationnever exceeds the target concentration. The production cells areperfused for a period of time necessary to obtain an α-Gal A preparationwith between 50-70%, preferably 60%, of the total glycans beingsialylated.

Increasing Circulating Half-Life of Serum α-Gal A by PEGylation of α-GalA

Also according to this invention, the circulatory half-life of a humanglycosylated α-Gal A preparation is enhanced by complexing α-Gal A withpolyethylene glycol. Poly(ethylene glycol) (PEG) is a water solublepolymer that when covalently linked to proteins, alters their propertiesin ways that extend their potential uses. Polyethylene glycolmodification (“PEGylation”) is a well established technique which hasthe capacity to solve or ameliorate many of the problems of protein andpeptide pharmaceuticals.

The improved pharmacological performance of PEG-proteins when comparedwith their unmodified counterparts prompted the development of this typeof conjugate as a therapeutic agent. Enzyme deficiencies for whichtherapy with the native enzyme was inefficient (due to rapid clearanceand/or immunological reactions) can now be treated with equivalentPEG-enzymes. For example, PEG-adenosine deaminase has already obtainedFDA approval. Delgado et al., Crit. Rev. Ther. Drug Carrier Syst. 9:249-304 (1992).

The covalent attachment of PEG to α-galactosidase from green coffeebeans alters the catalytic properties of the enzyme by masking specificdeterminant sites on the molecule. This results in an increase in K_(m)and a decrease in V_(max) values against p-nitrophenyl substrateanalogs. Wieder & Davis, J. Appl. Biochem. 5: 337-47 (1983).α-galactosidase was still able to cleave terminal galactose residuesfrom human saliva blood group substance B. Antibody and lectin-specificbinding were lost from PEG-α-galactosidase. Antibodies generated fromnative α-galactosidase can block enzyme activity, and this inhibition isgradually lost when tested against preparations of the enzyme withprogressively higher amounts of PEG. By contrast, antisera from animalsimmunized with PEG-α-galactosidase did not inhibit enzyme activity inany α-galactosidase or PEG-α-galactosidase preparation. These resultsindicate that PEG tends to cover lectin-specific carbohydrate moietiesand antigenic determinants and that these sites probably remain crypticduring in vivo processing of PEG-enzymes.

Covalent attachment of PEG to proteins requires activation of thehydroxyl terminal group of the polymer with a suitable leaving groupthat can be displaced by nucleophilic attack of the ε-amino terminal oflysine and the α-amino group of the N-terminus. Several chemical groupshave been exploited to activate PEG. For each particular application,different coupling methods provide distinct advantages. Differentmethods of PEGylation have a surprising and dramatic impact on factorssuch as retention of bioactivity, stability and immunogenicity of theresulting PEGylated proteins and peptides. Francis et al., Int. J.Hematol. 68(1): 1-18 (1998). For example, a linkerless PEGylationtechnique attaches only PEG to the target molecule. More specifically,the application of a biologically optimized PEGylation technique, usingtresyl monomethoxy PEG (TMPEG), to a variety of target proteins reveals,as described by Francis et al., Int. J. Hematol. 68(1): 1-18 (1998), anexceptional ability to conserve biological activity of the target. This,and the benefit of adding nothing other than PEG (which has been shownto be safe for use in human therapeutics), to the protein makes themethod ideal for the modification of α-Gal A.

Four possible sites for coupling PEG to proteins are the (1) aminogroups (N-terminus and lysine); (2) carboxyl groups (aspartic acid andglutamic acid); (3) sulfhydryl groups (cysteine); and (4) carbohydrategroups (aldehydes generated after periodate treatment). Coupling to thecarboxyl groups of proteins and to aldehyde groups on carbohydratesrequires a PEG reagent with a nucleophilic amino group. This chemistrychanges the pI of α-Gal A after the negatively charged carboxyl groupsare bound by PEG. Any changes in pI may affect the biological activityof α-Gal A. Furthermore, coupling PEG to the carbohydrate chains mayaffect uptake of α-Gal A by the M6P receptor, which is critical forbiological activity. Sulfhydryl chemistry also affects the physicalstructure of the molecule and is not recommended.

Commonly used methods for PEGylation form an amide bond between theamino groups of a protein and the methoxy group on monomethoxy-PEG.NHS-PEG is commercially available and results in an amide bond betweenthe protein and PEG. However amide bond formation changes pI due to theloss of the positive charge of the —NH₂ group.

A method for coupling PEG to α-Gal A without affecting its pI usestresyl-PEG. Tresyl-PEG couples through amino groups and form a stablesecondary amine. Secondary amines offer the advantage of retaining thepositive charge of the amino group. The tresyl-PEG reagent iscommercially available and is stable as a lyophilized and desiccatedpowder. Tresyl-PEG has been thoroughly characterized and the reactionand by-products are well understood. Accordingly, in a preferredembodiment, the α-Gal A preparation is complexed using tresylmonomethoxy PEG (TMPEG) to form a PEGylated-α-Gal A. The PEGylated-α-GalA is then purified to provide an isolated, PEGylated-α-Gal A.

α-Gal A contains 18 amino groups, 17 ε-amino groups (lysine) and oneα-amino group (N-terminus). The reaction can be controlled to produceα-Gal A with minimal substitutions and then molecules with one PEG permolecule, or a lesser mean number of PEG moieties per molecule, can bepurified from the unsubstituted and multiply substituted forms. Multiplesubstitutions on α-Gal A may not significantly affect biologicalactivity; therefore the final product may consist of a heterogeneousmixture of one to 18 attached PEG molecules. The level of substitutionwill depend on the level of retained enzymatic activity. It should benoted that a decrease in enzymatic activity can be offset by an enhancedtherapeutic effect derived from lengthening the circulatory half-lifeand reducing immune recognition of α-Gal A. Thus, in developing aPEG-α-Gal A product, the ratio of PEG to α-Gal A should be dependent onbiological activity, and not solely on enzymatic activity.

The PEGylation reaction requires a controlled pH, buffer composition,and protein concentration. Proper reaction conditions can be achieved byan ultrafiltration/diafiltration step, which is currently used in themanufacturing process. Immediately before reacting, tresyl-PEG isquickly solubilized in water with continuous stirring. This solution isthen added to the prepared α-Gal A and allowed to react for a controlledamount of time and at a controlled temperature (e.g., 2 hours at 250°C.). PEGylation can occur prior to the final purification process, whichwill eliminate adding steps to the purification procedure. After thecoupling is complete, PEG-α-Gal A is processed by the remaining steps ofthe purification process. Performing the reaction before the Q column(anion exchange) allows for two purification steps to remove thereaction byproducts. Since PEG does not contain any negative charge, itwill not be retained by the Q Sepharose®, and will elute in the voidvolume.

The amount of PEGylation can be measured by known techniques. Forexample, fluorescamine fluoresces when bound to α-amino and ε-aminogroups of proteins. The percent loss in fluorescence after PEGylationcorrelates to the percentage of PEG bound to α-Gal A. Pierce's BCA assayfor total protein can be used to determine protein concentration. Themethylumbelliferyl-α-D-galactopyranoside (4-MUF-α-Gal) activity assay isused to evaluate the effect of PEG-α-Gal A enzymatic activity. α-Gal Acontains M6P, which is required for uptake into lysosomes. Interferencefrom PEG on M6P receptor recognition can be evaluated using a cell-basedassay to monitor cellular uptake of PEG-α-Gal A into lysosomes.

Methods of Administration of α-Gal A Preparation

Compositions of the present invention (i.e., comprising various α-Gal Aglycoforms) may be administered by any route which is compatible withthe α-Gal A preparation. The purified α-Gal A preparation can beadministered to individuals who produce insufficient or defective α-GalA protein or who may benefit from α-Gal A therapy. Therapeuticpreparations of the present invention may be provided to an individualby any suitable means, directly (e.g., locally, as by injection,implantation or topical administration to a tissue locus) orsystemically (e.g., orally or parenterally).

The route of administration may be oral or parenteral, includingintravenous, subcutaneous, intra-arterial, intraperitoneal, ophthalmic,intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral,intradermal, intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal,transdermal, or via inhalation. Intrapulmonary delivery methods,apparatus and drug preparation are described, for example, in U.S. Pat.Nos. 5,785,049, 5,780,019, and 5,775,320, each incorporated herein byreference. A preferred method of intradermal delivery is byiontophoretic delivery via patches; one example of such delivery istaught in U.S. Pat. No. 5,843,015, which is incorporated herein byreference.

A particularly useful route of administration is by subcutaneousinjection. An α-Gal A preparation of the present invention is formulatedsuch that the total required dose may be administered in a singleinjection of one or two milliliters. In order to allow an injectionvolume of one or two milliliters, an α-Gal A preparation of the presentinvention may be formulated at a concentration in which the preferreddose is delivered in a volume of one to two milliliters, or the α-Gal Apreparation may be formulated in a lyophilized form, which isreconstituted in water or an appropriate physiologically compatiblebuffer prior to administration. Subcutaneous injections of α-Gal Apreparations have the advantages of being convenient for the patient, inparticular by allowing self-administration, while also resulting in aprolonged plasma half-life as compared to, for example, intravenousadministration. A prolongation in plasma half-life results inmaintenance of effective plasma α-Gal A levels over longer time periods,the benefit of which is to increase the exposure of clinically affectedtissues to the injected α-Gal A and, as a result, increase the uptake ofa α-Gal A into such tissues. This allows a more beneficial effect to thepatient and/or a reduction in the frequency of administration.Furthermore, a variety of devices designed for patient convenience, suchas refillable injection pens and needle-less injection devices, may beused with the α-Gal A preparations of the present invention as discussedherein.

Administration may be by periodic injections of a bolus of thepreparation, or may be administered by intravenous or intraperitonealadministration from a reservoir which is external (e.g., an IV bag) orinternal (e.g., a bioerodable implant, a bioartificial organ, or apopulation of implanted α-Gal A production cells). See, e.g., U.S. Pat.Nos. 4,407,957 and 5,798,113, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the α-Gal Apreparation described above can administered in these methods.

The route of administration and the amount of protein delivered can bedetermined by factors that are well within the ability of skilledartisans to assess. Furthermore, skilled artisans are aware that theroute of administration and dosage of a therapeutic protein may bevaried for a given patient until a therapeutic dosage level is obtained.

Pharmaceutical Formulation of α-Gal A Protein

This invention further provides novel formulations of an α-Gal Apreparation that are substantially free of non-α-Gal A proteins, such asalbumin, non-α-Gal A proteins produced by the host cell, or proteinsisolated from animal tissue or fluid.

The preparation preferably comprises part of an aqueous orphysiologically compatible fluid suspension or solution. The carrier orvehicle is physiologically compatible so that, in addition to deliveryof the desired preparation to the patient, it does not otherwiseadversely affect the patient's electrolyte and/or volume balance. Usefulsolutions for parenteral administration may be prepared by any of themethods well known in the pharmaceutical art. See, e.g., REMINGTON'SPHARMACEUTICAL SCIENCES (Gennaro, A., ed.), Mack Pub., 1990.Non-parenteral formulations, such as suppositories and oralformulations, can also be used.

Preferably the formulation contains an excipient. Pharmaceuticallyacceptable excipients for α-Gal A which may be included in theformulation are buffers such as citrate buffer, phosphate buffer,acetate buffer, and bicarbonate buffer, amino acids, urea, alcohols,ascorbic acid, phospholipids; proteins, such as serum albumin, collagen,and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomes;polyvinylpyrollidone; sugars, such as dextran, mannitol, sorbitol, andglycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000,PEG-6000); glycerol; glycine or other amino acids; and lipids. Buffersystems for use with α-Gal A preparations include citrate; acetate;bicarbonate; and phosphate buffers (all available from Sigma). Phosphatebuffer is a preferred embodiment. A preferred pH range for α-Gal Apreparations is pH 4.5-7.4.

The formulation also preferably contains a non-ionic detergent.Preferred non-ionic detergents include Polysorbate 20, Polysorbate 80,Triton X-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octylβ-glucoside, Brij 35, Pluronic, and Tween 20 (all available from Sigma).

A particularly preferred formulation contains Polysorbate 20 orPolysorbate 80 non-ionic detergent and phosphate-buffered saline, mostpreferably at pH 6.

For lyophilization of α-Gal A preparations, the protein concentrationcan be 0.1-10 mg/mL. Bulking agents, such as glycine, mannitol, albumin,and dextran, can be added to the lyophilization mixture. In addition,possible cryoprotectants, such as disaccharides, amino acids, and PEG,can be added to the lyophilization mixture. Any of the buffers,excipients, and detergents listed above, can also be added.

In a preferred formulation α-Gal A for injection is at a concentrationof 1 mg/mL

Formulations for administration may include glycerol and othercompositions of high viscosity to help maintain the agent at the desiredlocus. Biocompatible polymers, preferably bioresorbable, biocompatiblepolymers (including, e.g., hyaluronic acid, collagen, polybutyrate,lactide, and glycolide polymers and lactide/glycolide copolymers) may beuseful excipients to control the release of the agent in vivo.Formulations for parenteral administration may include glycocholate forbuccal administration, methoxysalicylate for rectal administration, orcutric acid for vaginal administration. Suppositories for rectaladministration may be prepared by mixing an α-Gal A preparation of theinvention with a non-irritating excipient such as cocoa butter or othercompositions that are solid at room temperature and liquid at bodytemperatures.

Formulations for inhalation administration may contain lactose or otherexcipients, or may be aqueous solutions which may containpolyoxyethylene-9-lauryl ether, glycocholate or deoxycocholate. Apreferred inhalation aerosol is characterized by having particles ofsmall mass density and large size. Particles with mass densities lessthan 0.4 gram per cubic centimeter and mean diameters exceeding 5 μmefficiently deliver inhaled therapeutics into the systemic circulation.Such particles are inspired deep into the lungs and escape the lungs'natural clearance mechanisms until the inhaled particles deliver theirtherapeutic payload. (Edwards et al., Science 276: 1868-1872 (1997)).α-Gal A preparations of the present invention can be administered inaerosolized form, for example by using methods of preparation andformulations as described in U.S. Pat. Nos. 5,654,007, 5,780,014, and5,814,607, each incorporated herein by reference. Formulation forintranasal administration may include oily solutions for administrationin the form of nasal drops, or as a gel to be applied intranasally.

Formulations for topical administration to the skin surface may beprepared by dispersing the α-Gal A preparation with a dermatologicalacceptable carrier such as a lotion, cream, ointment, or soap.Particularly useful are carriers capable of forming a film or layer overthe skin to localize application and inhibit removal. For topicaladministration to internal tissue surfaces, the α-Gal A preparation maybe dispersed in a liquid tissue adhesive or other substance known toenhance adsorption to a tissue surface. For example, several mucosaladhesives and buccal tablets have been described for transmucosal drugdelivery, such as in U.S. Pat. Nos. 4,740,365, 4,764,378, and 5,780,045,each incorporated herein by reference. Hydroxypropylcellulose orfibrinogen/thrombin solutions may also be incorporated. Alternatively,tissue-coating solutions, such as pectin-containing formulations may beused.

The preparations of the invention may be provided in containers suitablefor maintaining sterility, protecting the activity of the activeingredients during proper distribution and storage, and providingconvenient and effective accessibility of the preparation foradministration to a patient. An injectable formulation of an α-Gal Apreparation might be supplied in a stoppered vial suitable forwithdrawal of the contents using a needle and syringe. The vial would beintended for either single use or multiple uses. The preparation canalso be supplied as a prefilled syringe. In some instances, the contentswould be supplied in liquid formulation, while in others they would besupplied in a dry or lyophilized state, which in some instances wouldrequire reconstitution with a standard or a supplied diluent to a liquidstate. Where the preparation is supplied as a liquid for intravenousadministration, it might be provided in a sterile bag or containersuitable for connection to an intravenous administration line orcatheter. In preferred embodiments, the preparations of the inventionare supplied in either liquid or powdered formulations in devices whichconveniently administer a predetermined dose of the preparation;examples of such devices include a needle-less injector for eithersubcutaneous or intramuscular injection, and a metered aerosol deliverydevice. In other instances, the preparation may be supplied in a formsuitable for sustained release, such as in a patch or dressing to beapplied to the skin for transdermal administration, or via erodibledevices for transmucosal administration. In instances where thepreparation is orally administered in tablet or pill form, thepreparation might be supplied in a bottle with a removable cover. Thecontainers may be labeled with information such as the type ofpreparation, the name of the manufacturer or distributor, theindication, the suggested dosage, instructions for proper storage, orinstructions for administration.

Dosages for Administration of α-Gal A Preparation

The present invention further provides methods for administering anα-Gal A preparation to a patient with Fabry disease, atypical variant ofFabry disease or any condition in which a reduced level or mutant formof α-Gal A is present. The dose of administration is preferably 0.05-5.0mg, more preferably between 0.1-0.3 mg, of the α-Gal A preparation perkilogram body weight and is administered weekly or biweekly. In apreferred embodiment, a dose of about 0.2 mg/kg is administeredbiweekly. Regularly repeated doses of the protein are necessary over thelife of the patient. Subcutaneous injections can be used to maintainlonger term systemic exposure to the drug. The subcutaneous dosage canbe between 0.01-10.0 mg, preferably 0.1-5.0 mg, of the α-Gal Apreparation per kg body weight biweekly or weekly. Dosages of α-Gal Apreparations that are administered by intramuscular injections may bethe same or different than those injected subcutaneously; in a preferredembodiment, intramuscular dosages are smaller and administered lessfrequently. The α-Gal A preparation can also be administeredintravenously, e.g., in a intravenous bolus injection, in a slow pushintravenous injection, or by continuous intravenous injection.Continuous IV infusion (e.g., over 2-6 hours) allows the maintenance ofspecific levels in the blood.

An alternative preferred method for administering an α-Gal A preparationto a patient involves administering a preferred dose of an α-Gal Apreparation weekly or biweekly for a period of several years, e.g., upto three years, during which time a patient is monitored clinically toevaluate the status of his or her disease. Clinical improvement measuredby, for example, improvement in renal or cardiac function or patient'soverall well-being (e.g., pain), and laboratory improvement measured by,for example, reductions in urine, plasma, or tissue CTH levels, may beused to assess the patient's health status. In the event that clinicalimprovement is observed after this treatment and monitoring period, thefrequency of α-Gal A administration may be reduced. For example, apatient receiving weekly injections of an α-Gal A preparation may changeto biweekly injections. Alternatively, a patient receiving biweeklyinjections of an α-Gal A preparation may switch to monthly injections.Following such a change in dosing frequency, the patient should bemonitored for another several years, e.g., a three year period, in orderto assess Fabry disease-related clinical and laboratory measures. In apreferred embodiment, the administered dose does not change if a changein dosing frequency is made. This ensures that certain pharmacokineticparameters (e.g. maximal plasma concentration [C_(max)], time to maximalplasma concentration [t_(max)], plasma, half-life [t_(1/2)], andexposure as measured by area under the curve [AUC]) remain relativelyconstant following each administered dose. Maintenance of thesepharmacokinetic parameters will result in relatively constant levels ofreceptor-mediated uptake of α-Gal A into tissues as dose frequencieschange.

A patient with atypical variant of Fabry disease, e.g., exhibitingpredominantly cardiovascular abnormalities or renal involvement, istreated with these same dosage regiments, i.e., from 0.05 mg/kg to 5mg/kg weekly or biweekly. The dose is adjusted as needed. For example, apatient with the cardiac variant phenotype who is treated withα-galactosidase A enzyme replacement therapy will have a change in thecomposition of their heart and improved cardiac function followingtherapy. This change can be measured with standard echocardiographywhich is able to detect increased left ventricular wall thickness inpatients with Fabry disease (Goldman et al., J Am Coll Cardiol 7:1157-1161 (1986)). Serial echocardiographic measurements of leftventricular wall thickness can be conducted during therapy, and adecrease in ventricular wall size is indicative of a therapeuticresponse. Patients undergoing α-gal A enzyme replacement therapy canalso be followed with cardiac magnetic resonance imaging (MRI). MRI hasthe capability to assess the relative composition of a given tissue. Forexample, cardiac MRI in patients with Fabry disease reveals depositedlipid within the myocardium compared with control patients (Matsui etal., Am Heart J 117; 472-474. (1989)). Serial cardiac MRI evaluations ina patient undergoing enzyme replacement therapy can reveal a change inthe lipid deposition within a patient's heart. Patients with the renalvariant phenotype can also benefit from α-galactosidase A enzymereplacement therapy. The effect of therapy can be measured by standardtests of renal function, such as 24-hour urine protein level, creatinineclearance, and glomerular filtration rate. The following Examples arepresented in order to more fully illustrate the preferred embodiments ofthe invention. These Examples should in no way be construed as limitingthe scope of the invention, as defined by the appended claims.

Example 1 Preparation and Use of Constructs Designed to Deliver andExpress α-Gal A

Two expression plasmids, pXAG-16 and pXAG-28, were constructed. Theseplasmids contain human α-Gal A cDNA encoding the 398 amino acids of theα-Gal A enzyme (without the α-Gal A signal peptide); the human growthhormone (hGH) signal peptide genomic DNA sequence, which is interruptedby the first intron of the hGH gene; and the 3′ untranslated sequence(UTS) of the hGH gene, which contains a signal for polyadenylation.Plasmid pXAG-16 has the human cytomegalovirus immediate-early (CMV IE)promoter and first intron (flanked by non-coding exon sequences), whilepXAG-28 is driven by the collagen Iα2 promoter and exon 1, and alsocontains the β-actin gene's 5′ UTS, which contains the first intron ofthe β-actin gene.

1.1 Cloning of the Complete α-Gal A cDNA, and Construction of the α-GalA Expression Plasmid pXAG-16

The human α-Gal cDNA was cloned from a human fibroblast cDNA librarythat was constructed as follows. Poly-A⁺ mRNA was isolated from totalRNA, and cDNA synthesis was performed using reagents for the lambdaZapII® system according to the manufacturer's instructions (StratageneInc., LaJolla, Calif.). Briefly, “first strand” cDNA was generated byreverse transcription in the presence of an oligo-dT primer containingan internal XhoI restriction endonuclease site. Following treatment withRNase H, the cDNA was nick-translated with DNA polymerase I to generatedouble stranded cDNA. This cDNA was made blunt-ended with T4 DNApolymerase, and ligated to EcoRI adaptors. The products of this ligationwere treated with T4 DNA kinase and digested with XhoI. The cDNA wasfractionated by Sephacryl®-400 chromatography. Large and medium sizefractions were pooled and the cDNAs ligated to EcoRI and XhoI-digestedLambda ZapII arms. The products of this ligation were then packaged andtitered. The primary library had a titer of 1.2×10⁷ pfu/mL and anaverage insert size of 925 bp.

A 210 bp probe from exon 7 of the human α-Gal A gene (FIG. 1, SEQ IDNO:1) was used to isolate the cDNA. The probe itself was isolated fromgenomic DNA by the polymerase chain reaction (PCR) using the followingoligonucleotides: 5′-CTGGGCTGTAGCTATGATAAAC-3′ (Oligo 1; SEQ ID NO:6)and 5′-TCTAGCTGAAGCAAAACAGTG-3′ (Oligo 2; SEQ ID NO:7). The PCR productwas then used to screen the fibroblast cDNA library, and positive cloneswere isolated and further characterized. One positive clone, phage 3A,was subjected to the lambda ZapII® system excision protocol (Stratagene,Inc., La Jolla, Calif.), according to the manufacturer's instructions.This procedure yielded plasmid pBSAG3A, which contains the α-Gal A cDNAsequence in the pBluescriptSK-™ plasmid backbone. DNA sequencingrevealed that this plasmid did not contain the complete 5′ end of thecDNA sequence. Therefore, the 5′ end was reconstructed using a PCRfragment amplified from human genomic DNA. To accomplish this, a 268 bpgenomic DNA fragment (FIG. 2, SEQ ID NO:2) was amplified using thefollowing oligonucleotides: 5′-ATTGGTCCGCCCCTGAGGT-3′ (Oligo 3; SEQ IDNO:8) and 5′-TGATGCAGGAATCTGGCTCT-3′ (Oligo 4; SEQ ID NO:9). Thisfragment was subcloned into a “TA” cloning plasmid (Invitrogen Corp.,San Diego, Calif.) to generate plasmid pTAAGEI. Plasmid pBSAG3A, whichcontains the majority of the α-Gal A cDNA sequence, and pTAAGEI, whichcontains the 5′ end of the α-Gal A cDNA, were each digested with SacIIand NcoI. The positions of the relevant SacII and NcoI sites within theamplified DNA fragment are shown in FIG. 2. The 0.2 kb SacII-NcoIfragment from pTAAGEI was isolated and ligated to equivalently digestedpBSAG3A. This plasmid, pAGAL, contains the complete α-Gal A cDNAsequence, including the sequence encoding the α-Gal A signal peptide.The cDNA was completely sequenced (shown in FIG. 3 including the α-Gal Asignal peptide; SEQ ID NO:3) and found to be identical to the publishedsequence for the human α-Gal A cDNA (Genbank sequence HUMGALA).

The plasmid pXAG-16 was constructed via several intermediates, asfollows. First, pAGAL was digested with SacII and XhoI and blunt-ended.Second, the ends of the complete α-Gal A cDNA were ligated to XbaIlinkers and subcloned into XbaI digested pEF-BOS (Mizushima et al.,Nucl. Acids Res. 18: 5322, 1990), creating pXAG-1. This constructcontains the human granulocyte-colony stimulating factor (G-CSF) 3′ UTSand the human elongation factor-1a (EF-1a) promoter flanking the cDNAencoding α-Gal A plus the α-Gal A signal peptide, such that the 5′ endof the α-Gal A cDNA is fused to the EF-1a promoter. To create aconstruct with the CMV IE promoter and first intron, the α-Gal A cDNAand G-CSF 3′ UTS were removed from pXAG-1 as a 2 kb XbaI-BamHI fragment.The fragment was blunt-ended, ligated to BamHI linkers, and insertedinto BamHI digested pCMVflpNeo (which was constructed as describedbelow). The orientation was such that the 5′ end of the α-Gal A cDNA wasfused to the CMV IE promoter region.

pCMVflpNeo was created as follows. A CMV IE gene promoter fragment wasamplified by PCR using CMV genomic DNA as a template and theoligonucleotides: 5′-TTTTGGATCCCTCGAGGACATTGATTATTGACTAG-3′ (SEQ IDNO:10) and 5′-TTTTGGATCCCGTGTCAAGGACGGTGAC-3′ (SEQ ID NO:11). Theresulting product (a 1.6 kb fragment) was digested with BamHI, yieldinga CMV promoter-containing fragment with cohesive BamHI-digested ends.The neo expression unit was isolated from plasmid pMC1neopA (StratageneInc., La Jolla, Calif.) as a 1.1 kb XhoI-BamHI fragment. The CMVpromoter-containing and neo fragments were inserted into a BamHI-,XhoI-digested plasmid (pUC12). Notably, pCMVflpNeo contains the CMV IEpromoter region, beginning at nucleotide 546 and ending at nucleotide2105 (of Genbank sequence HS5MIEP), and the neomycin resistance genedriven by the Herpes Simplex Virus (HSV) thymidine kinase promoter (theTKneo gene) immediately 5′ to the CMV IE promoter fragment. Thedirection of transcription of the neo gene is the same as that of theCMV promoter fragment. This intermediate construct was called pXAG-4.

To add the hGH 3′ UTS, the GCSF 3′ UTS was removed from pXAG-4 as anXbaI-SmaI fragment and the ends of pXAG-4 were made blunt. The hGH 3′UTS was removed from pXGH5 (Selden et al., Mol. Cell. Biol. 6:3173-3179, 1986) as a 0.6 kb SmaI-EcoRI fragment. After blunt-endingthis fragment, it was ligated into pXAG-4 immediately after theblunt-ended XbaI site of pXAG-4. This intermediate was called pXAG-7.The TKneo fragment was removed from this plasmid as a HindIII-ClaIfragment and the ends of the plasmid were blunted by “filling-in” withthe Klenow fragment of DNA polymerase I. A neomycin resistance genedriven by the SV40 early promoter was ligated in as a blunted ClaI-BsmBIfragment from a digest of pcDNeo (Chen et al., Mol. Cell. Biol. 7:2745-2752, 1987), placing the nco transcription unit in the sameorientation as the α-Gal A transcription unit. This intermediate wascalled pXAG-13.

To complete pXAG-16, which has the 26 amino acid hGH signal peptidecoding sequence and first intron of the hGH gene, a 2.0 kb EcoRI-BamHIfragment of pXAG-13 was first removed. This fragment included the α-GalA cDNA and the hGH 3′ UTS. This large fragment was replaced with 3fragments. The first fragment consisted of a 0.3 kb PCR product ofpXGH5, which contains the hGH signal peptide coding sequence andincludes the hGH first intron sequence, from a synthetic BamHI sitelocated just upstream of the Kozak consensus sequence to the end of thehGH signal peptide coding sequence. The following oligonucleotides wereused to amplify this fragment (Fragment 1): 5′-TTTTGGATCCACCATGGCTA-3′(Oligo HGH101; SEQ ID NO:12) and 5′-TTTTGCCGGCACTGCCCTCTTGAA-3′ (OligoHGH102; SEQ ID NO:13). The second fragment consisted of a 0.27 kb PCRproduct containing sequences corresponding to the start of the cDNAencoding the 398 amino acid α-Gal A enzyme (i.e., lacking the α-Gal Asignal peptide) to the NheI site. The following oligonucleotides wereused to amplify this fragment (Fragment 2):5′-TTTTCAGCTGGACAATGGATTGGC-3′ (Oligo AG10; SEQ ID NO:14) and5′-TTTTGCTAGCTGGCGAATCC-3′ (Oligo AG 11; SEQ ID NO:15). The thirdfragment consisted of the NheI-EcoRI fragment of pXAG-7 containing theremaining α-Gal A sequence as well as the hGH 3′ UTS (Fragment 3).

Fragment 1 (digested with BamHI and NaeI), Fragment 2 (digested withPvuII and NheI), and Fragment 3 were mixed with the 6.5 kb BamHI-EcoRIfragment of pXAG-13 containing the neo gene and the CMV IE promoter andligated together to generate plasmid pXAG-16 (FIG. 4).

1.2 Construction of the α-Gal A Expression Plasmid pXAG-28

The human collagen Iα2 promoter was isolated for use in the α-Gal Aexpression construct pXAG-28 as follows. A 408 bp PCR fragment of humangenomic DNA containing part of the human collagen Iα2 promoter wasisolated using the following oligonucleotides:

(Oligo 72; SEQ ID NO: 16) 5′-TTTTGGATCCGTGTCCCATAGTGTTTCCAA-3′ and(Oligo 73; SEQ ID NO: 17) 5′-TTTTGGATCCGCAGTCGTGGCCAGTACC-3′.

This fragment was used to screen a human leukocyte library in EMBL3(Clontech Inc., Palo Alto, Calif.). One positive clone (phage 7H)containing a 3.8 kb EcoRI fragment was isolated and cloned into pBSIISK+(Stratagene Inc., La Jolla, Calif.) at the EcoRI site (creatingpBS/7H.2). An AvrII site was introduced in pBSIISK+ by digesting withSpeI, which cleaves within the pBSIISK+ polylinker, “filling-in” withthe Klenow fragment of DNA polymerase I, and inserting theoligonucleotide 5′-CTAGTCCTAGGA-3′ (SEQ ID NO:18). This variant ofpBSIISK+ was digested with BamHI and AvrII and ligated to the 121 bpBamHI-AvrII fragment of the original 408 bp collagen Iα2 promoter PCRfragment described above, creating pBS/121COL.6.

The plasmid pBS/121COL.6 was digested with XbaI, which cleaves withinthe pBSIISK+ polylinker sequence, “filled-in” with the Klenow fragmentof DNA polymerase I, and digested with AvrII. The 3.8 kb BamHI-AvrIIfragment of pBS/7H.2 was isolated and the BamHI site made blunt-ended bytreatment with Klenow enzyme. The fragment was then digested with AvrIIand ligated to the AvrII-digested vector, thus creating the collagenpromoter plasmid pBS/121 bpCOL7H.18.

Next the collagen promoter was fused to the 5′ UTS of the human β-actingene, which contains the first intron of the human β-actin gene. Toisolate this sequence, a 2 kb PCR fragment was isolated from humangenomic DNA using the following oligonucleotides:

(Oligo BA1; SEQ ID NO: 19) 5′-TTTTGAGCACAGAGCCTCGCCT-3′ and(Oligo BA2; SEQ ID NO: 20) 5′-TTTTGGATCCGGTGAGCTGCGAGAATAGCC-3′.

This fragment was digested with BamHI and BsiHKAI to release a 0.8 kbfragment containing the β-actin 5′ UTS and intron. A 3.6 kb SaII-SrfIfragment was then isolated from the collagen promoter plasmidpBS/121bpCOL7H.18 as follows. pBS/121 bpCOL7H.18 was partially digestedwith BamHI (the BamHI site lies at the 5′ end of the collagen Iα2promoter fragment), made blunt-ended by treatment with the Klenowfragment, and ligated to a SalI linker (5′-GGTCGACC-3′), thereby placinga SalI site upstream of the collagen Iα2 promoter. This plasmid was thendigested with SalI and SrfI (the SrfI site lies 110 bp upstream of thecollagen Iα2 promoter CAP site), and the 3.6 kb fragment was isolated.The 0.8 and 3.6 kb fragments were combined with SalI- and BamHI-digestedpBSIISK- (Stratagene Inc., La Jolla, Calif.), and a fragment composed ofthe following four oligonucleotides annealed together (forming afragment with a blunt end and a BsiHKAI end):

(Oligo COL-1; SEQ ID NO: 21)5′-GGGCCCCCAGCCCCAGCCCTCCCATTGGTGGAGGCCCTTTTGGAGGCACCCTAGGGCCAGGAAACTTTTGCCGTAT-3′, (Oligo COL-2; SEQ ID NO: 22)5′-AAATAGGGCAGATCCGGGCTTTATTATTTTAGCACCACGGCCGCCGAGACCGCGTCCGCCCCGCGAGCA-3′, (Oligo COL-3; SEQ ID NO: 23)5′-TGCCCTATTTATACGGCAAAAGTTTCCTGGCCCTAGGGTGCCTCCAAAAGGGC CTCCACCAATGGGAGGGCTGGGGCTGGGGGCCC-3′, and(Oligo COL-4; SEQ ID NO: 24)5′-CGCGGGGCGGACGCGGTCTCGGCGGCCGTGGTGCTAAAATAATAAAG CCCGGATC-3′.

These four oligonucleotides, when annealed, correspond to the regionbeginning at the SrfI site of the collagen promoter and continuingthrough the BsiHKAI site of the β-actin promoter. The resulting plasmidwas designated pCOL/β-actin.

To complete the construction of pXAG-28, the SalI-BamHI fragment ofpCOL/β-actin, containing the collagen Iα2 promoter and β-actin 5′ UTS,was isolated. This fragment was ligated to two fragments from pXAG-16(see Example 1.1 and FIG. 4): (1) the 6.0 kb BamHI fragment (containingthe neo gene, plasmid backbone, the cDNA encoding the 398 amino acidα-Gal A enzyme, and the hGH 3′ UTS); and (2) the 0.3 kb BamHI-XhoIfragment (which contains the SV40 poly A sequence from pcDneo). pXAG-28contains the human collagen Iα2 promoter fused to the human β-actin 5′UTS, the hGH signal peptide (which is interrupted by the hGH firstintron), the cDNA encoding the α-Gal A enzyme, and the hGH 3′ UTS. A mapof the completed expression construct pXAG-28 is shown in FIG. 5.

1.3 Transfection and Selection of Fibroblasts Electroporated with α-GalA Expression Plasmids

In order to express α-Gal A in fibroblasts, secondary fibroblasts werecultured and transfected according to published procedures (Selden etal., WO 93/09222).

The plasmids pXAG-13, pXAG-16 and pXAG-28 were transfected byelectroporation into human foreskin fibroblasts to generate stablytransfected clonal cell strains, and the resulting α-Gal A expressionlevels were monitored as described in Example 1.4. Secretion of α-Gal Aby normal foreskin fibroblasts is in the range of 2-10 units/10⁶cells/24 hours. In contrast, the transfected fibroblasts displayed meanexpression levels as shown in Table 2.

TABLE 2 Mean α-Gal A expression levels (± standard deviation) pXAG-13:420 ± 344 U/10⁶ cells/day N = 26 clonal strains (range 3-1133 U/10⁶cells/day) pXAG-16: 2,051 ± 1253 U/10⁶ cells/day N = 24 clonal strains(range 422-5200 U/10⁶ cells/day) pXAG-28: 141 ± 131 U/10⁶ cells/day N =38 clonal strains (range 20-616 U/10⁶ cells/day)

These data show that all three expression constructs are capable ofincreasing α-Gal A expression many times that of nontransfectedfibroblasts. Expression by fibroblasts stably transfected with pXAG-13,which encodes α-Gal A linked to the α-Gal A signal peptide, wassubstantially lower than expression by fibroblasts transfected withpXAG-16, which differs only in that the signal peptide is the hGH signalpeptide, the coding sequence of which is interrupted by the first intronof the hGH gene.

Each time the transfected cells were passaged, the secreted α-Gal Aactivity was determined, the cells were counted, and the cell densitywas calculated. Based on the number of cells harvested and the timeallowed for secretion of α-Gal A, the specific expression rate of α-GalA was determined and is reported in Tables 3 and 4 as secreted units (ofα-Gal A) per 10⁶ cells per 24 hour period. Cell strains desirable forgene therapy or for use in generation of material for purification ofα-Gal A should display stable growth and expression over severalpassages. Data from the cell strains shown in Tables 3 and 4, which werestably transfected with the α-Gal A expression construct pXAG-16,illustrate the fact that α-Gal A expression is stably maintained duringserial passage.

TABLE 3 Growth and Expression of BRS-11 Cells Containing the α-Gal AExpression Construct pXAG-16 Expression Cell Density Passage (units/10⁶cells/24 hr) (cells/cm²) 13 2601 4.80 × 10⁴ 14 1616 4.40 × 10⁴ 15 35954.40 × 10⁴

TABLE 4 Growth and Expression of HF503-242 Cells Containing the α-Gal AExpression Construct PxAG-16 Expression Cell Density Passage (units/10⁶cells/24 hr) (cells/cm²) 5 4069 2.80 × 10⁴ 6 7585 3.55 × 10⁴ 7 5034 2.48× 10⁴

1.4 Quantification of α-Gal A Expression

The activity of α-Gal A activity was measured using the water-solublesubstrate 4-methylumbelliferyl-α-D-galactopyranoside (4-MUF-gal;Research Products, Inc.) by a modification of the protocol described byIoannou et al., J. Cell Biol. 119: 1137-1150 (1992). The substrate wasdissolved in substrate buffer (0.1 M citrate-phosphate, pH 4.6) to aconcentration of 1.69 mg/mL (5 mM). Typically, 10 mL of culturesupernatant was added to 75 mL of the substrate solution. The tubes werecovered and allowed to incubate in a 37° C. water bath for 60 minutes.At the end of the incubation period, 2 mL of glycine-carbonate buffer(130 mM glycine, 83 mM sodium carbonate, at pH 10.6), were used to stopthe reaction. The relative fluorescence of each sample was measuredusing a model TK0100 fluorometer (Hoefer Scientific Instruments) whichhas a fixed excitation wavelength of 365 nm and detects a fixed emissionwavelength of 460 nm. The readings of the samples were compared tostandards prepared from a 1 mM stock of methylumbelliferone (SigmaChemical Co.), and the amount of hydrolyzed substrate was calculated.The activity of α-Gal A is expressed in units; one unit of α-Gal Aactivity is equivalent to one nanomole of substrate hydrolyzed per hourat 37° C. Cell expression data were generally expressed as units ofα-Gal A activity secreted/10⁶ cells/24 hours. This assay was also usedto measure the amount of α-Gal activity in cell lysates and in samplesfrom various α-Gal purification steps, as discussed below.

1.5 Preparation of Gene-Activated α-Gal A (GA-GAL)

Production of gene-activated α-Gal A (GA-GAL) occurred by insertion ofregulatory and structural DNA sequences upstream of the human α-Gal Acoding sequence, using the GA technology substantially as described inU.S. Pat. No. 5,733,761, herein incorporated by reference. The preciseinsertion of the gene-activating sequence occurs as a result ofhomologous recombination between DNA present on a transfected DNAfragment and genomic DNA sequences upstream of the α-Gal A locus in ahuman cell. The gene-activating sequence itself contains α-Gal A codingsequence up to, but not including, the signal peptide cleavage site.Cells containing an activated α-Gal A locus were isolated and subjectedto drug selection to isolate cells with increased GA-GAL production.

A targeting DNA fragment containing an appropriate gene-activatingsequence was introduced into host human cell lines by electroporation.One such cell line is HT-1080, a certified cell line available from ATCC(Rockville, Md.). The gene activation plasmid (targeting construct)pGA213C containing such a DNA fragment is shown in FIG. 9. This plasmidcontains sequences designed to activate a portion of the endogenousα-Gal A locus in the host cell line, and contains sequences encoding thesignal peptide, but not human α-Gal A. The targeting construct alsocontains expression cassettes for the bacterial neo and mouse dhfrgenes. These allow for the selection of stably integrated targetingfragments (via the neo gene) and for subsequent selection of the dhfrgene using step-wise methotrexate (MTX) selection.

In addition, pGA213C contains sequences designed to target chromosomalsequences upstream of the endogenous α-Gal A locus by homologousrecombination. Homologous recombination between the endogenous α-Gal Alocus and the 9.6 kb DNA fragment of pGA213C is shown in FIG. 10.

pGA213C was constructed to delete 962 bp of genomic sequences extendingfrom positions −1183 to −222 relative to the methionine initiation codonof α-Gal A, upon homologous recombination of the pGA213C fragment withthe X-chromosomal α-Gal A locus. Transcriptional activation of the α-GalA locus occurs through precise targeting of the exogenous regulatorysequences upstream of the α-Gal A coding region. The resulting GA-GALlocus cause transcription to initiate from the CMV promoter and toproceed through CMV exon 1, the aldolase intron and the seven exons andsix introns of the α-Gal A coding sequence. Splicing of the largeprecursor mRNA joins the exogenous CMV exon (inserted by targeting) withthe entire endogenous first exon of α-Gal A transcript. Translation ofthe GA-GAL mRNA results in pre GA-GAL with a thirty one amino acidsignal peptide. Upon secretion from the host cell, the signal peptide isremoved. Correctly targeted cell lines are first identified bypolymerase chain reaction screening for the presence of the GA-GAL mRNA.Clones producing the GA-GAL mRNA are also found to secrete enzymaticallyactive α-Gal A into the culture media. Subsequent confirmation oftargeting events is accomplished by restriction enzyme digestion andSouthern blot hybridization analysis of genomic DNA.

Cells were exposed to stepwise methotrexate (“MTX”) selection. Followingselection in 0.05 μM MTX, a clone of cells was isolated and subjected to0.1 μM MTX selection. From this process a pool of cells resistant to 0.1μM MTX was isolated (cell line RAG001), expanded in culture andcharacterized.

Example 2 α-Gal A Purification

The following is a preferred method for producing, purifying, andtesting α-Gal A. The purification process maintains α-Gal A in asoluble, active, native form throughout the purification process. Theprotein is not exposed to extremes of pH, organic solvents ordetergents, is not proteolytically cleaved during the purificationprocess, and does not form aggregates. The purification process isdesigned not to alter the distribution of α-Gal A glycoforms.

2.1 Purification of α-Gal A

Example 2.1 illustrates that α-Gal A may be purified to near-homogeneityfrom the conditioned medium of cultured human cell strains that havebeen stably transfected to produce the enzyme. α-Gal A is isolated fromα-Gal A containing media using a series of five chromatographic steps.The five steps utilize various separation principles which takeadvantage of different physical properties of the enzyme to separateα-Gal A from contaminating material. Included are hydrophobicinteraction chromatography on butyl Sepharose®, ionic interaction onhydroxyapatite, anion exchange chromatography on Q Sepharose®, and sizeexclusion chromatography on Superdex® 200. In addition to being thefinal step in the purification process, size exclusion chromatographyalso serves as an effective means to exchange the purified protein intoa formulation-compatible buffer.

A. Use of Butyl Sepharose® Chromatography as a First Step in thePurification of α-Gal A

Cold conditioned medium (1.34 liters) was clarified by centrifugationand filtered through a 0.45 μm cellulose acetate filter using glassfiber prefilters. While stirring, the pH of the cold, filtered mediumwas adjusted to 5.6 bp the dropwise addition of 1 N HCl, and ammoniumsulfate was added to a final concentration of 0.66 M by the dropwiseaddition of a stock solution (room temperature) of 3.9 M ultrapureammonium sulfate. The medium was stirred for an additional 5 minutes at4° C., filtered as before, and applied to a Butyl Sepharose® 4 Fast Flowcolumn (81 ml column volume, 2.5×16.5 cm; Pharmacia, Uppsala, Sweden)that had been equilibrated in 10 mM MES-Tris, pH 5.6, containing 0.66 Mammonium sulfate (buffer A). The chromatography was performed at 4° C.on a Gradi-Frac™ System (Pharmacia, Uppsala, Sweden) equipped within-line UV (280 nm) and conductivity monitors for assessing totalprotein and salt concentration, respectively. After sample applicationat a flow rate of 10 ml/min, the column was washed with 10 columnvolumes of buffer A. The α-Gal A was eluted from the Butyl Sepharose®column with a 14 column volume linear gradient from buffer A (containingammonium sulfate) to 10 mM MES-Tris, pH 5.6 (no ammonium sulfate).Fractions were assayed for α-Gal A activity by the 4-MUF-gal assay, andthose containing appreciable enzyme activity were pooled. As seen inFIG. 8 and the purification summary (Table 5), this step removesapproximately 99% of the contaminating protein (pre-column sample=8.14 gtotal protein; post-column sample-0.0638 g total protein).

TABLE 5 Purification of α-Gal A from the Conditioned Medium of StablyTransfected Human Fibroblasts α-Gal A Specific Fold Purification VolumeActivity Total Prote Activity Purification Percent Step (ml) (×10⁶Units) in (mg) (×10⁶ Units/mg) (Cumulative) Recovery Culture 1340 14.68140 0.0018 =1 =100 supernatant Buty 417 14.1 63.8 0.221 123 96.6Sepharose ® Heparin 134 12.1 14.6 0.829 436 82.9 Sepharose ® Hydroxy- 479.73 4.46 2.18 1220 66.6 apatite Q 31.5 8.91 3.31 2.69 1503 61.0Sepharose ® Superdex ® 10 8.58 2.93 2.92 1634 59.0 200 B. Use of HeparinSepharose ® Chromatography as a Step for Purification of α-Gal A

The Butyl Sepharose® column peak fractions were dialyzed at 4° C.against (4 liters) of 10 mM MES-Tris, pH 5.6 (changed once). Theconductivity of the dialysate was adjusted to 1.0 mMHO at 4° C. byaddition of H₂O or NaCl as necessary. Afterward, the sample was appliedto a column of Heparin Sepharose® 6 Fast Flow (Pharmacia, Uppsala,Sweden; 29 ml column volume, 2.5×6 cm) that had been equilibrated in 10mM MES-Tris, pH 5.6, containing 9 mM NaCl (buffer B). This was done at4° C. at a flow rate of 10 ml/min. In-line UV (280 nm) and conductivitymonitors measured total protein and salt concentration. After the samplewas applied, the column was washed with 10 column volumes of buffer Bfollowed by a 3 column volume linear gradient to 8% buffer C/92% bufferB (where buffer C is 10 mM MES-Tris, pH 5.6, containing 250 mM NaCl) anda 10 column volume wash with 8% buffer C. This was followed by elutionof α-gal A with a 1.5 column volume linear gradient to 29% buffer C anda subsequent 10 column volume linear gradient to 35% buffer C. Fractionswere assayed for α-gal A activity, and those containing appreciableactivity were pooled.

C. Use of Hydroxyapatite Chromatography as a Step for Purification ofα-Gal A

The heparin pool was filtered and applied directly to a column ofCeramic Hydroxyapatite HC (40 μm; American International Chemical,Natick, Mass.; 12 ml column volume, 1.5×6.8 cm) that had beenequilibrated in 1 mM sodium phosphate, pH 6.0 (buffer D). Thechromatography was performed at room temperature on a hybridGradi-Frac™/FPLC® System (Pharmacia, Uppsala, Sweden) equipped within-line UV (280 nm) and conductivity monitors. After the sample wasapplied (5 ml/min), the column was washed with 10 column volumes ofbuffer D. The α-Gal A was eluted with a 7 column volume linear gradientto 42% buffer E/58% buffer D (where buffer E is 250 mM sodium phosphate,pH 6.0) followed by a 10 column volume gradient to 52% buffer E.Fractions were assayed for α-Gal A activity, and the fractionscontaining appreciable activity were pooled.

D. Use of Q Sepharose® Anion Exchange Chromatography as a Step forPurification of α-Gal A

The hydroxyapatite pool was diluted approximately 1.5 fold with H₂O to afinal conductivity of 3.4-3.6 mMHO at room temperature. After filtering,the sample was applied to a column of Q Sepharose® HP (Pharmacia,Uppsala, Sweden; 5.1 ml column volume, 1.5×2.9 cm) equilibrated in 10%buffer G/90% buffer F, where buffer F is 25 M sodium phosphate, pH 6.0,and buffer G is 25 mM sodium phosphate, pH 6.0, 250 mM NaCl. Thechromotography was performed at room temperature on theGradi-Frac™/FPLC® hybrid system (Pharmacia, Uppsala, Sweden), and totalprotein and salt concentrations were monitored by the in-line monitors.The sample was applied at a flow rate of 5 ml/min, then the followingsteps were performed: (1) a 5 column volume wash at 10% buffer G, (2) a7 column volume wash at 12% buffer G, (3) a 3 column volume lineargradient to 50% buffer G, (4) a 10 column volume linear gradient to 53%buffer G, (5) a 3 column volume gradient to 100% buffer G, and (6) a 10column volume wash at 100% buffer G. The α-Gal A eluted primarily duringsteps 3 and 4. Fractions containing appreciable activity were pooled(the “Q pool”).

E. Use of Superdex®-200 Gel Filtration Chromatography as a Step forPurification of α-Gal A

The Q pool was concentrated approximately 5-fold using Centriprep®-10centrifugal concentrator units (Amicon, Beverly, Mass.), and applied toa column of Superdex® 200 (Pharmacia, Uppsala, Sweden; 189 ml columnvolume, 1.6×94 cm). The column was equilibrated and eluted with 25 mMsodium phosphate, pH 6.0, containing 150 mM NaCl. The chromatography wasperformed on an FPLC® system (Pharmacia, Uppsala, Sweden) at roomtemperature using an in-line UV monitor (280 nm) to follow elution ofthe protein. The volume of the sample applied to the column was ≦2 ml,the flow rate was 0.5 ml/min, and the fraction size was 2 ml. Multiplecolumn runs were performed; fractions were assayed for α-Gal A activityand fractions containing appreciable activity were pooled.

The pooled fractions from the Superdex® 200 column were concentratedusing Centriprep 10 units, aliquoted, snap-frozen, and stored at −80° C.for short periods of time. A summary of this example of α-Gal Apurification is shown in Table 5. The final yield of α-Gal A was 59% ofthe starting material activity, and the specific activity of thepurified product was 2.92×10⁶ units/mg protein. The resulting productshowed a high level of purity after electrophoresis under reducingconditions on a 4-15% SDS-polyacrylamide gel, which was subsequentlysilver-stained.

Summary

The purification process provides highly purified α-Gal A. The majorityof the purification occurs in the first 2 steps of the process, whilethe final three steps serve to polish the material by removing theremaining minor contaminants. The last step, size exclusionchromatography on Superdex® 200, also serves to exchange the α-Gal Ainto a formulation-compatible buffer.

2.2 Size of α-Gal A Produced by Stably Transfected Human Cells inCulture

The structural and functional properties of purified human α-Gal A wereinvestigated. The resulting product showed a high level of purity afterelectrophoresis under reducing conditions on a 4-15% SDS-polyacrylamidegel, which was subsequently silver-stained.

The molecular mass of α-Gal A was estimated by MALDI-TOF massspectrometry. These results demonstrate that the molecular mass of thedimer is 102,353 Da, while that of the monomer is 51,002 Da. Theexpected molecular mass of the monomer, based on amino acid composition,is 45,400 Da. Therefore, the carbohydrate content of the enzyme accountsfor up to 5,600 Da of the molecular weight.

2.3 Carbohydrate Modification of α-Gal A Produced by Stably TransfectedHuman Cells

The glycosylation pattern of α-Gal A produced in accordance with theinvention was also evaluated. Proper glycosylation is important foroptimal in vivo activity of α-Gal A; α-Gal A expressed innon-glycosylating systems is inactive or unstable. Hantzopolous et al.,Gene 57: 159 (1987). Glycosylation is also important for theinternalization of α-Gal A into the desired target cells, and affectsthe circulating half-life of the enzyme in vivo. On each subunit ofα-Gal A, there are four sites available for addition ofasparagine-linked carbohydrate chains, of which only three are occupied.Desnick et al., In THE METABOLIC AND MOLECULAR BASES OF INHERITEDDISEASE, (McGraw Hill, New York, 1995) pp 2741-2780.

A sample of α-Gal A produced by stably transfected cells was treatedwith neuraminidase, which is isolated from A. urafaciens,(Boehringer-Mannheim, Indianapolis, Ind.) to remove sialic acid. Thisreaction was performed by treating 5 mg of α-Gal A overnight with 10 mUof neuraminidase at room temperature in a total volume of 10 mL ofacetate buffered saline (ABS, 20 mM sodium acetate, pH. 5.2, 150 mMNaCl).

Purified α-Gal A produced by stably transfected cells was alsodephosphorylated using alkaline phosphatase (calf intestinal alkalinephosphatase, Boehringer-Mannheim, Indianapolis, Ind.), by treating 5 mgof α-Gal A overnight at room temperature with 15 U of alkalinephosphatase in ABS (pH raised to 7.5 with 1 M Tris).

The samples were analyzed by SDS-PAGE and/or isoelectric focusingfollowed by Western blotting with an anti-α-Gal A-specific antibody. Theantibody used was a rabbit polyclonal anti-peptide antibody, which wasproduced using a peptide representing amino acids 68-81 of α-Gal A as animmunogen. Following transfer of the protein to PVDF (Millipore,Bedford, Mass.), the membrane was probed with a 1:2000 dilution of theanti-serum in 2.5% blotto (non-fat dry milk in 20 mM Tris-HCl, pH 7.5,0.05% Tween-20). This was followed by detection with goat anti-rabbitIgG conjugated to horseradish peroxidase (Organo Technique/Cappella,Durham, N.C.; 1:5000 dilution) and reagents of the ECL chemiluminescencekit (Amersham, Arlington Heights, Ind.).

Treatment of α-Gal A with neuraminidase followed by SDS-PAGE analysisresulted in a shift in molecular mass (approximately 1500-2000 Da or 4-6sialic acids/monomer), suggesting that there is extensive modificationof α-Gal A with sialic acid. For reference, the plasma form of α-Gal Ahas 5-6 sialic acid residues per monomer, and the placental form has0.5-1.0 sialic acid residues per monomer. Bishop et al., J. Biol. Chem.256: 1307 (1981).

Another method used to examine the sialic acid and M6P modifications ofα-Gal A was isoelectric focusing (IEF), where the samples are separatedon the basis of their isoelectric point (pI) or net charge. Thus,removal of charged residues such as sialic acid or phosphate from α-GalA would be expected to alter the mobility of the protein in the IEFsystem.

To perform the IEF experiment, samples of α-Gal A produced in accordancewith the invention were treated with neuraminidase and/or alkalinephosphatase, mixed 1:1 with 2× Novex sample buffer (with 8 M urea, pH3.0-7.0), and loaded onto a 6 M urea IEF gel (5.5% polyacrylamide) madeusing Pharmalyte® (Pharmacia, Uppsala, Sweden; pH 3.0-6.5; Pharmalyte®4-6.5 and 2.5-5.5, 0.25 mL each per gel). Isoelectric point standards(Bio-Rad) were also included. Following electrophoresis, the gel wastransferred to PVDF, and Western blot analysis performed as describedabove.

Neuraminidase treatment of the enzyme increased the pI of all threeisoforms, indicating that all were modified to some extent by sialicacid. These data suggest that the α-Gal A preparations produced asdescribed herein should have a desirable plasma half-life, indicatingthat this material is well suited for pharmacological use. Further,treatment of neuraminidase-treated α-Gal A with alkaline phosphatasefurther increased the pI of a portion of the protein to approximately5.0-5.1, indicating that the enzyme bears one or more M6P residues. Thismodification is required for efficient internalization of α-Gal A by thetarget cells.

The N-linked carbohydrate chains of α-Gal A were analyzed byion-exchange HPLC (Glyco-Sep C) and labeling of the non-reducing endwith the fluorescent compound 2-amino benzamide (AB). The results of theanalysis of AB-glycans from three separate α-Gal A preparations aresummarized in Table 6. All three preparations had a Z number greaterthan 170. Further, over 67% of the glycans were sialylated, over 16% ofthe glycans were phosphorylated, and less than 16% were neutral. Theseresults compared very favorably compared to results reported in theprior art. For example, Desnick et al., (U.S. Pat. No. 5,356,804)reported that over 60% of the glycans were neutral, with only 11% beingsialylated.

TABLE 6 Results of Analysis of AB-glycans from GA-GAL % Treatment Znumber Neutral % Mono- % Di- % Tri- % Tetra- None 170.04 16.83 22.839.45 15.34 5.58 None 177.71 14.22 20.63 44.62 14.2 6.31 None 171.6815.81 20.73 43.2 14.33 5.39 Mean (N = 3) 173.14 15.62 21.39 42.42 14.625.76 Neuraminidase 24.36 85.25 5.14 9.61 ND ND Alk. Phosphatase 150.9323.38 24.47 34.28 13.58 4.29 GA-GALpreparations of the present Desnicket al., Percent of Total: invention U.S. Pat. No. 5,356,804 TotalP-glycans 16.62 24.1 Total Sialylated 67.57 11 Total Neutral 15.62 62.9(hih-mannose and hybrid)

Further detailed characterizations of the purified GA-GAL preparationsare provided in Table 7.

TABLE 7 GA-GAL Purified Bulk Assay 40-173-KH 42-202-KH Specific activity2.75  2.80 SDS-PAGE Coomassie   100%  100% SDS-PAGE Silver stain  99.6% 100% Reverse phase HPLC   100% 99.94 Size exclusion    0% 0.01%chromatography Internalization by foreskin 123.6% 94.3% fibroblasts

2.4 Increasing Proportion of Charged α-Gal A by Fractionation of α-Gal ASpecies

As discussed above, fractionation of α-Gal A glycoforms can occur atvarious steps in the purification process as described herein. In thepresent example, α-Gal A glycoforms were fractionated by size and bycharge. It is also possible to fractionate α-Gal A by a combination ofthese or other chromatographic techniques as described above.

For size fractionation of α-Gal A glycoforms, size exclusionchromatography was performed on a Superdex® 200 column (Pharmacia, 1.6cm by 94.1 cm) equilibrated in phosphate buffered saline at pH 6. α-GalA (2.6 mg in 1 mL) was loaded onto the column, and the column was elutedat 0.35 mL/min. Fractions were collected across the elution profile, andthe fractions comprising the broad elution peak of α-Gal A were analyzedby SDS-PAGE, then visualized with silver stain. The fractions at theleading edge of the peak contained α-Gal A of the highest molecularweight, and as the fractions continued across the peak, the apparentmolecular weight of the α-Gal A gradually decreased. Fractions of α-GalA were then selected and pooled to provide α-Gal A preparation of thedesired molecular weight ranges.

For fractionation of α-Gal A glycoforms by charge, α-Gal A wasfractionated by Q-Sepharose® chromatography. The Q-Sepharose® column(1.5 cm by 9.4 cm) was equilibrated in 20 mM sodium phosphate, pH 6.0,containing 30 mM NaCl and the flow rate was maintained at 5 mL/min.α-Gal A in (130 mg in 166 mL) was loaded onto the column, washed withequilibration buffer then eluted with 20 mM sodium phosphate, pH 6.0,containing 130 mM NaCl. For more extensive fractionation, a gradientelution (e.g., 10 column volumes) from the equilibration buffer to theelution buffer can be used. Fractions were collected across the elutionprofile, and the fractions comprising the elution peak of α-Gal A wereanalyzed by SDS-PAGE, then visualized by silver stain. The lowestmolecular weight species observed on the gel eluted in the wash and atthe leading edge of the peak, the highest molecular weight glycoformseluted towards the end of the peak. The lower molecular weight speciescorrespond to the less negatively charged glycoforms of α-Gal A, whichbind less tightly to the positively charged Q-Sepharose® column(comprised of a quaternary amine substituted resin). The α-Gal A speciesof highest negative charge eluted later in the elution profile and havea higher molecular weight, as analyzed by SDS-PAGE. The fractionation bycharge was confirmed by isoelectric focusing of the eluted fractions orof selected pools.

Thus, both the fractionation by size and the fractionation by chargepermitted the selection of highly charged glycoforms of α-Gal A.

2.5 Mannose or Mannose-6-Phosphate (M6P) Mediated Internalization ofα-Gal A

For the α-Gal A produced by stably transfected cells to be an effectivetherapeutic agent for α-Gal A deficiencies, the enzyme must beinternalized by the affected cells. α-Gal A is minimally active atphysiological pH levels, for example, in the blood or interstitialfluids. α-Gal A metabolizes accumulated lipid substrates optimally onlywhen internalized in the acidic environment of the lysosome. Thisinternalization is mediated by the binding of α-Gal A to M6P receptors,which are expressed on the cell surface and deliver the enzyme to thelysosome via the endocytic pathway. The M6P receptor is ubiquitouslyexpressed; most somatic cells express M6P to some extent. The mannosereceptor, which is specific for exposed mannose residues onglycoproteins, is less prevalent. The mannose receptors are generallyfound only on macrophage and macrophage-like cells, and provide anadditional means of α-Gal A entry into these cell types.

In order to demonstrate M6P-mediated internalization of α-Gal A, skinfibroblasts from a Fabry disease patient (NIGMS Human Genetic MutantCell Repository) were cultured overnight in the presence of increasingconcentrations of purified α-Gal A of the invention. Some of the samplescontained 5 mM soluble M6P, which competitively inhibits binding to andinternalization by the M6P receptor. Other samples contained 30 mg/mLmannan, which inhibits binding to and internalization by the mannosereceptor. Following incubation, the cells were washed and harvested byscraping into lysis buffer (10 mM Tris, pH 7.2, 100 mM NaCl, 5 mM EDTA,2 mM Pefabloc™ (Boehringer-Mannheim, Indianapolis, Ind.) and 1% NP-40).The lysed samples were then assayed for protein concentration and α-GalA activity. The results are expressed as units of α-Gal A activity/mgcell protein. The Fabry cells internalized α-Gal A in a dose-dependentmanner. This internalization was inhibited by M6P, but there was noinhibition with mannan. Therefore, internalization of α-Gal A in Fabryfibroblasts is mediated by the M6P receptor, but not by the mannosereceptor.

α-Gal A is also internalized in vitro by endothelial cells, importanttarget cells for the treatment of Fabry disease. Human umbilical veinendothelial cells (HUVECs) were cultured overnight with 7500 units ofα-Gal A; some of the wells contained M6P. After the incubation period,cells were harvested and assayed for α-Gal A as described above. Thecells incubated with α-Gal A had enzyme levels almost 10-fold those ofcontrol (no incubation with α-Gal A) cells. M6P inhibited theintracellular accumulation of α-Gal A, suggesting that theinternalization of α-Gal A by HUVECs is mediated by the M6P receptor.Thus, the human α-Gal A of the invention is internalized by clinicallyrelevant cells.

Few cultured human cell lines are known to express the mannose receptor.However, a mouse macrophage-like cell line (J774.E) which bears mannosereceptors but few if any M6P receptors can be used to determine whetherpurified α-Gal A of the invention is internalized via the mannosereceptor. Diment et al., J. Leukocyte Biol. 42: 485-490 (1987). J774.Ecells were cultured overnight in the presence of 10,000 units/mL α-GalA. Selected samples also contained 2 mM M6P, and others contained 100mg/mL mannan. The cells were washed and harvested as described above,and the total protein and α-Gal A activity of each sample wasdetermined. M6P does not inhibit the uptake of α-Gal A by these cells,while mannan decreases the accumulated α-Gal A levels by 75%. Thus, theα-Gal A of the invention may be internalized by the mannose receptor incell types that express this particular cell surface receptor.

Example 3 Pharmaceutical Formulation

Preparation of Buffer Solutions and Formulations

α-Gal A Purified Bulk is diluted to final concentration with α-Gal ADiluent. Based on the volume of purified bulk to be formulated, theconcentration of α-Gal A (mg/mL), and the desired concentration of α-GalA in the final formulation, the volume of α-Gal A diluent required isdetermined. α-Gal A diluent is prepared within 24 hours of use by mixingappropriate quantities of WFI, sodium chloride, and sodium phosphatemonobasic, and adjusting the pH to 6.0 with sodium hydroxide solution.The composition of α-Gal A Diluent is listed in Table 8.

TABLE 8 COMPOSITION OF α-GAL A DILUENT (per Liter) Component Part NumberQuantity Sodiumchloride(USP) 100-1916 8.8 g Sodium hydroxide, 5N200-1903 qs to adjust pH to 6.0 Sodium phosphate, 100-1913 3.5 gmonobasic (USP) Water for Injection (USP) 100-2301 qs ad 1.0 L

One liter or smaller volumes of α-Gal A Diluent are filtered by vacuumfiltration using sterile 0.2 mm nylon filters (Nalge Nunc International,Rochester, N.Y.). Larger volumes are filtered by positive pressure usinga peristaltic pump and 0.2 mm Supor® capsule filters (Pall, PortWashington, N.Y.). All filters are subjected to post-filtration bubblepoint integrity testing. Mixing and filtration steps are performed in acertified Class 100 laminar flow hood. α-Gal A diluent is added to α-GalA purified bulk in a mixing vessel to give a 1 mg/ml final solution.Then, the appropriate volume of polysorbate 20 (Tween 20, Spectrum) isadded to reach a final concentration of 0.02%.

Example 4 Desialylated Degalactosylated α-Gal A

To explore the effect of glycosylation on the biodistribution of α-GalA, a purified preparation of α-Gal A was sequentially deglycosylated andeach form injected into mice. The organs of the mice were collected atfour hours post-injection and immunohistochemistry on the tissuesperformed to visualize possible changes in the biodistribution of theprotein.

The α-Gal A was first treated with neuraminidase (sialidase) to removesialic acid residues, leaving galactose moieties exposed. A portion ofthis sialidase-treated was further reacted with β-galactosidase toremove galactose residues; this left N-acetylglucosamine (GlcNAC)residues exposed. The GlcNACs were then removed byN-acetylglucosaminidase, leaving the core mannose groups on the protein.Untreated α-Gal A (control) or one of the treated forms of the proteinwere injected via the tall vein into mice. Four hours after theinjections, the liver, spleen, heart, kidney and lungs from the micewere collected, preserved, and immunostained for detection of α-Gal A.

When compared to control animals receiving untreated protein, micereceiving the sialidase treated enzyme (galactose residues exposed) hadmore α-Gal A localized in the liver and correspondingly less of theenzyme in other examined organs. Additionally, the staining pattern inthe liver was quite different. In control animals, the α-Gal A localizedto primarily the Kupffer cells and endothelial cells with only moderatehepatocyte staining. In animals receiving the sialidase treated α-Gal A,the enzyme localized only to the hepatocytes, consistent with the knownbiodistribution of the asialoglycoprotein receptor. This effect ofdeglycosylation on the biodistribution was reversed when the galactoseresidues were removed by β-galactosidase. The staining pattern observedin the liver of the mice receiving this protein without galactosemoieties was similar to that of the control animals; the majority of thestaining was in Kupffer cells and endothelial cells, with minimalhepatocyte staining. Further treatment of the α-Gal A withN-acetylglucosaminidase did not alter the staining pattern from thatobserved for the β-galactosidase treated protein; that is, removal ofthe N-acetylglucosamine residues seemed to have little effect on thebiodistribution of α-Gal A.

Example 5 Correction of Fabry Fibroblasts by Human FibroblastsExpressing α-Gal A

For gene therapy, an implant of autologous cells producing α-Gal A mustproduce the enzyme in a form modified appropriately to “correct” theα-Gal A deficiency in target cells. To assess the effect of α-Gal Aproduction by transfected human fibroblasts on Fabry cells, fibroblastsharvested from Fabry disease patients (NIGMS Human Genetics Mutant CellRepository) were co-cultured with an α-Gal A production cell strain(BRS-11) in Transwells® (Costar, Cambridge, Mass.). Fabry cells werecultured in 12-well tissue culture dishes, some of which containedinserts (Transwells®, 0.4 mm pore size) having a surface on which cellscan be grown. The growth matrix of the insert is porous and allowsmacromolecules to pass from the upper to the lower milieu. One set ofinserts contained normal human foreskin (HF) fibroblasts, which secreteminimal levels of α-Gal A, while another set contained the stablytransfected human fibroblast strain, BRS-11, which secretes largeamounts of α-Gal A. In the wells co-cultured with α-Gal A productioncells, α-Gal A can enter the medium bathing the Fabry cells, andpotentially be internalized by the Fabry cells.

The data in Table 9 show that Fabry cells internalized the secretedα-Gal A. The intracellular levels of α-Gal A were monitored for 3 days.Those cells cultured alone (no insert) or in the presence ofnon-transfected foreskin fibroblasts (HF insert) had very lowintracellular levels of α-Gal A activity. The Fabry cells cultured withthe α-Gal A production (BRS-11 insert) cells, however, exhibited enzymelevels similar to those of normal cells by the end of Day 2 (normalfibroblasts have 25-80 units α-Gal A/mg protein). That the correction isattributable to α-Gal A taken up via the M6P receptor is demonstrated bythe inhibition with M6P (BRS-11 insert+M6P).

TABLE 9 CORRECTION OF FABRY FIBROBLASTS BY HUMAN FIBROBLASTS EXPRESSINGα-Gal A ACTIVITY (units/mg total protein) no HF BRS-11 BRS-11 Timeinsert insert insert insert + M6P Day 1 2 ± 1 2 ± 1 13 ± 1  4 ± 1 Day 22 ± 1 2 ± 1 40 ± 11 6 ± 2 Day 3 2 ± 1 5 ± 1 85 ± 1  9 ± 1

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto. In the specificationand the appended claims, the singular forms include plural references,unless the context clearly dictates otherwise. All patents andpublications cited in this specification are incorporated by reference.

1-45. (canceled)
 46. A human α-galactosidase A (α-Gal A) glycoproteinpreparation, wherein the preparation comprises an average of about 1 to2 mannose-6-phosphate residues per α-Gal A monomer and greater than 50%of the total glycans of the preparation are complex-type glycans. 47.The preparation of claim 46, wherein the preparation is purified to atleast 98% homogeneity, as measured by SDS-PAGE or reverse phase HPLC.48. The preparation of claim 46, wherein the preparation has a specificactivity of at least 2.0×10⁶ units/mg protein.
 49. The preparation ofclaim 46, wherein the preparation comprises an average of two complexglycans per α-Gal A monomer.
 50. The preparation of claim 46, whereinmore than 50% of the total glycans of the preparation are sialyated. 51.The preparation of claim 46, wherein the complex glycans have about 2 to4 sialic acid residues.
 52. The preparation of claim 46, wherein about15% of the total glycans of the preparation are neutral glycans.
 53. Thepreparation of claim 46, wherein about 16% of the total glycans of thepreparation are phosphorylated glycans.
 54. The preparation of claim 46,wherein the preparation is isolated from human cells transfected with anα-Gal A/mammalian expression vector construct.
 55. A pharmaceuticalcomposition comprising a human α-galactosidase A (α-Gal A) glycoproteinpreparation, wherein the preparation comprises an average of about 1 to2 mannose-6-phosphate residues per α-Gal A monomer and greater than 50%of the total glycans of the preparation are complex-type glycans. 56.The composition of claim 55, wherein the composition comprises one unitdose between 0.1-0.3 mg of the preparation per kilogram body weight. 57.The composition of claim 55, wherein the preparation is purified to atleast 98% homogeneity, as measured by SDS-PAGE or reverse phase HPLC.58. The composition of claim 55, wherein the preparation has a specificactivity of at least 2.0×10⁶ units/mg protein.
 59. The composition ofclaim 55, wherein the preparation comprises an average of two complexglycans per α-Gal A monomer.
 60. The composition of claim 55, whereinmore than 50% of the total glycans of the preparation are sialyated. 61.The composition of claim 55, wherein the complex glycans have about 2 to4 sialic acid residues.
 62. The composition of claim 55, wherein about15% of the total glycans of the preparation are neutral glycans.
 63. Thecomposition of claim 55, wherein about 16% of the total glycans of thepreparation are phosphorylated glycans.
 64. The composition of claim 55,wherein the preparation is isolated from human cells transfected with anα-Gal A/mammalian expression vector construct.
 65. A method of treatinga subject, comprising administering to a subject in need thereof a humanα-galactosidase A (α-Gal A) glycoprotein preparation, wherein thepreparation comprises an average of about 1 to 2 mannose-6-phosphateresidues and greater than 50% of the total glycans of the preparationare complex-type glycans.
 66. The method of claim 65, wherein thepreparation is administered at a dose between 0.1-0.3 mg per kilogrambody weight.
 67. The method of claim 65, wherein the preparation isadministered weekly or biweekly.
 68. The method of claim 65, wherein thepreparation is purified to at least 98% homogeneity, as measured bySDS-PAGE or reverse phase HPLC.
 69. The method of claim 65, wherein thepreparation has a specific activity of at least 2.0×10⁶ units/mgprotein.
 70. The method of claim 65, wherein the preparation comprisesan average of two complex glycans per α-Gal A monomer.
 71. The method ofclaim 65, wherein more than 50% of the total glycans of the preparationare sialyated.
 72. The method of claim 65, wherein the complex glycanshave about 2 to 4 sialic acid residues.
 73. The method of claim 65,wherein about 15% of the total glycans of the preparation are neutralglycans.
 74. The method of claim 65, wherein about 16% of the totalglycans of the preparation are phosphorylated glycans.
 75. The method ofclaim 65, wherein the preparation is isolated from human cellstransfected with an α-Gal A/mammalian expression vector construct.